Synthesis and evaluation of [11C]Cimbi-806 as a potential PET ligand for 5-HT7 receptor imaging

Article abstract of DOI:10.1016/j.bmc.2012.05.005

2-(2′,6′-Dimethoxy-[1,1′-biphenyl]-3-yl)-N, N-dimethylethanamine has been identified as a potent ligand for the serotonin 7 (5-HT₇) receptor. In this study, we describe the synthesis, radiolabeling and in vivo evaluation of [¹¹C]2-(2

Full text of DOI:10.1016/j.bmc.2012.05.005

Bioorganic & Medicinal Chemistry 20 (2012) 4574–4581
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Bioorganic & Medicinal Chemistry
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Synthesis and evaluation of [11C]Cimbi-806 as a potential PET ligand
for 5-HT7 receptor imaging
Matthias M. Herth a,b,c, , Hanne D. Hansen a, , Anders Ettrup a, Agnete Dyssegaard a, Szabolcs Lehel c,
Jesper Kristensen b, Gitte M. Knudsen a,
⇑
a Center for Integrated Molecular Brain Imaging, Rigshospitalet and University of Copenhagen, Blegdamsvej 9, DK-2100 Copenhagen, Denmark
b Institute for Medicinal Chemistry, The Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark
c PET and Cyclotron Unit, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark
a r t i c l e i n f o
a b s t r a c t
2-(20,60-Dimethoxy-[1,10-biphenyl]-3-yl)-N,N-dimethylethanamine has been identified as a potent ligand
for the serotonin 7 (5-HT7) receptor. In this study, we describe the synthesis, radiolabeling and in vivo
evaluation of [11C]2-(20,60-dimethoxy-[1,10-biphenyl]-3-yl)-N,N-dimethylethanamine ([11C]Cimbi-806)
as a radioligand for imaging brain 5-HT7 receptors with positron emission tomography (PET). Precursor
and reference compound was synthesized and subsequent 11C-labelling with [11C]methyltriflate pro-
Article history:
Received 1 February 2012
Revised 6 April 2012
Accepted 3 May 2012
Available online 18 May 2012
duced [11C]Cimbi-806 in specific activities ranging from 50 to 300 GBq/
lmol. Following intravenous
Keywords:
injection, brain uptake and distribution of [11C]Cimbi-806 was assessed with PET in Danish Landrace pigs.
The time–activity curves revealed high brain uptake in thalamic and striatal regions (SUV ꢀ2.5) and
kinetic modeling resulted in distribution volumes (VT) ranging from 6 mL/cm3 in the cerebellum to
12 mL/cm3 in the thalamus. Pretreatment with the 5-HT7 receptor antagonist SB-269970 did not result
in any significant changes in [11C]Cimbi-806 binding in any of the analyzed regions. Despite the high
brain uptake and relevant distribution pattern, the absence of appropriate in vivo blocking with a
5-HT7 receptor selective compounds renders the conclusion that [11C]Cimbi-806 is not an appropriate
PET radioligand for imaging the 5-HT7 receptor in vivo.
[
11C]2-(20,60-dimethoxy-[1,10-biphenyl]-3-
yl)-N,N-dimethylethanamine
5-HT7 receptor
PET
[
11C]Cimbi-806
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
In vivo studies of cerebral 5-HT7 receptor binding in humans
would provide a significant advance in the understanding of the
The brain serotonin (5-HT) system is known to be involved in
various central nervous system (CNS) disorders, for example,
depression,1 and drugs with pharmacological effects on the 5-HT
system are among the most frequently used for brain disorders.
The most recently discovered receptor in the 5-HT receptor family,
the 5-HT7 receptor, is also considered associated with depression.
The atypical antipsychotic drug amisulpride has antidepressive
effects2,3 and studies in 5-HT7 receptor knock-out mice support
that the 5-HT7 receptor antagonism of amisulpride causes this
effect.4 Other atypical antipsychotics also have relative high affin-
ity for the 5-HT7 receptor although their direct involvement in alle-
viating depressive symptoms through 5-HT7 antagonism remains
to be investigated.5,6 Furthermore, there is also evidence that the
5-HT7 receptor is involved in psychosis, circadian rhythm, and
sleep architecture (for review see7).
above mentioned physiology and pathophysiology. Positron emis-
sion tomography (PET) is used to non-invasively quantify neurore-
ceptor binding in vivo and the availability of an appropriate PET
radiotracer for the 5-HT7 receptor would be of particular interest.
Previous attempts to develop a 5-HT7 receptor PET radioligand
have been made but unfortunately so far with limited success.8,9
Most recently, an 18F-labeled SB-269970 derivative was synthe-
sized and evaluated10,11 but in this study the arterial input function
was not determined and in the absence of a valid reference region,
the 5-HT7 receptor selectivity in vivo of this PET radioligand still
remains to be determined.
In the past years, several lead structures of 5-HT7 receptor bind-
ing ligands have been identified within different structural classes.12
Among these lead structures, biphenethylamines represent an easy
moiety for both organic synthesis and radiochemical labelling and in
addition has an interesting selectivity profile.13,14 Especially,
2-(20,60-dimethoxy-[1,10-biphenyl]-3-yl)-N,N-dimethylethanamine
(Cimbi-806) is of interest, because of its simple synthesis and its
favorable dissociation constant (Ki) for the 5-HT7 receptor
(Ki = 8.6 nM) over the 5-HT1A receptor (Ki = 4826 nM).13 This large
⇑
Corresponding author. Tel.: +45 3545 6720; fax: +45 3545 6713.
E-mail address: gmk@nru.dk (G.M. Knudsen).
Both authors have contributed equally.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.005

M. M. Herth et al. / Bioorg. Med. Chem. 20 (2012) 4574–4581
4575
difference in Ki is necessary because of the relative low 5-HT7 recep-
tor density (Bmax) in the brain and also because 5-HT7 and 5-HT1A
receptors are located in similar brain regions, such as the hippocam-
pus and cortical areas.15,16 Based on the literature values,15,16 one
can compute that in order to ensure that less than 10% PET signal
from the 5-HT1A receptors, a selectivity of at least 350-fold (based
on Bmax values in relevant regions) is required for specific imaging
the cerebral 5-HT7 receptor binding.
precursor (6) and the reference compound of [11C]Cimbi-806 were
synthesized in sufficient amounts in overall chemical yields of 43%
and 28%, respectively.
2.2. Lipophilicity
The lipophilicities of promising compounds were determined
using the HPLC method according to Krass et al.17 The logD of
Cimbi-806 was determined to be 4.4. This value may appear a little
high compared to the optimal logD interval for small molecules to
penetrate the blood–brain-barrier (BBB) as suggested by Rowley
et al.18 to be 2–3. But in our set-up other known CNS-PET ligands
(e.g., MDL 100907, altanserin or WAY 100635) show similar high
values. This fact gives rise to the assumption that [11C]Cimbi-806
may have similarly good properties for molecular imaging.
In this study, we synthesized the precursor for [11C]Cimbi-806
radiolabelling and subsequently evaluated its in vivo brain uptake
and binding characteristics in the pig.
2. Results and discussion
2.1. Organic chemistry
Bisphenylethylamines were synthesized a similar manner as
previously described.13 The method involves formation of 2,
6-dimethoxybenzeneboronic acid (4) and bromoethylamine deriv-
atives, which were consequently reacted via Suzuki-cross-coupling
to form the bisphenylethylamine matrix of [11C]Cimbi-806. The
Suzuki reaction was performed in good yields (>80%) under inert
conditions in a microwave in 2–4 h. Reaction times of the cross-cou-
pling varied and therefore, the completion of reaction was checked
by thin layer chromatography. However, conventional heating for
12 h lead mainly to the reduced phenethylamine derivative rather
than the bisphenylethylamine. Syntheses of precursor and refer-
ence compound of Cimbi-806 are summarized in Scheme 1.
To minimize synthesis efforts, both precursor and the reference
compound of [11C]Cimbi-806 was produced in an one-reaction
sequence. Thus, 3-bromophenethylamine was not directly bisme-
thylated, but Boc-protected which resulted in an almost complete
conversion. 2,6-dimethoxybenzeneboronic acid (4) was synthesized
in moderate yields (ꢀ30%) by lithiating 1,3-dimethoxybenzene and
then reacting with B(OMe)3. Afterwards, the Boc-protected deriva-
tive (1) and the boronic acid (4) were cross-coupled in excellent
yields (ꢀ90%).
2.3. In vitro autoradiography
The in vitro dissociation constant (Ki) for Cimbi-806 displace-
ment of [3H]SB-269970 binding on pig brain sections was
8.82 0.04 nM (Fig. 1), in accordance with the previously reported
Ki-value.13 The displacement of [3H]SB-269970 by Cimbi-806 was
encouraging for in vivo PET experiments.
2.4. Radiochemistry
Radiolabeling of [11C]Cimbi-806 (Scheme 2) using only 0.3 mg
precursor (6) and [11C]CH3OTf was done with a fully automated
system (RCY ꢀ95%).19
[
11C]Cimbi-806 at ꢀ400 s was the only
radioactive peak that was observed. Precursor (6) eluted 80 s prior
to the radiolabelled product as indicated by the UV-absorption
(data not shown). The radiosynthesis including HPLC purification
and formulation generated an injectable solution of [11C]Cimbi-
806 (radiochemical purity >96%) within 80–90 min. Typically,
0.6–1.8 GBq of [11C]Cimbi-806 was isolated with a specific activi-
ties (As) of 50–300 GBq/lmol at end of synthesis.
Several unsuccessful attempts were made to synthesize the pre-
cursor (6) from this cross-coupled intermediate (5). Reduction of
the carbamate to the mono-methylated secondary amine with
4 equiv of LiAlH failed and only trace amounts of the precursor
(6) could be detected via gas chromatography. In addition, methyl-
ation of (5) by activating the amine with NaH and reacting with
MeI followed by TFA Boc-deprotecting only resulted in primary
amine (8), probably since the alkylation was hindered by the
Boc-group. However, Boc-deprotection of (5) leads to yields close
to 80% and consequently reductive alkylation with aqueous form-
aldehyde resulted in the reference compound (Cimbi-806) in excel-
lent yields (ꢀ80%).
2.5. In vivo PET imaging
After intravenous injection of [11C]Cimbi-806, a rapid and high
uptake in the pig brain was observed (Fig. 2). Time-activity curves
(TACs) and summed PET images show highest [11C]Cimbi-806 up-
take in the thalamus and the striatum and lowest uptake in the
cerebellum. The TACs showed reversible kinetics of [11C]Cimbi-
806 with peak SUV at ꢀ20 min followed by a relative fast decline
in tissue activity.
With the high uptake of Cimbi-806 as seen in these experi-
ments, the relatively high lipophilicity value for Cimbi-806 does
not seem to be a problem. This outcome also holds true for many
other CNS-PET ligands (e.g., MDL 100907, altanserin, and WAY
100635) where high values are determined, but the tracers have
good brain uptake.
Two strategies were applied to synthesize the precursor: Mono-
methylated bromophenylethylamine was produced starting either
by reacting 3-bromophenethylbromide with aqueous methyl-
amine in a condensation reaction or by nosylation and methylation
of 3-bromophenethylamine in a one-pot reaction. The condensa-
tion reaction only resulted in poor amounts (ꢀ10%) of (2) even
when different bases or varying temperatures were applied. How-
ever, the Nosyl-protection followed by base activated methylation
with MeI resulted in complete conversion to (3).
Cross-coupling of (2) and (3) with 2,6-dimethoxybenzenebo-
ronic acid (4) under microwave yielded in the precursor (6) or
the intermediate (7) (ꢀ80–90%). The following thiolysis of (7) to
the precursor (6) succeeded in 52% without any attempts to opti-
mize this deprotection step, such as using higher concentration
of thiol groups or increasing the reaction temperature. We isolated
(6) in sufficient amounts for labelling purposes as well as for syn-
thesising Cimbi-806 via reductive alkylation. Taken together, the
To investigate the selectivity of [11C]Cimbi-806 in vivo, blocking
experiments was performed in three pigs after the baseline scan.
Pre-treatment followed by continuous infusion throughout the
scan with 0.5 or 1.0 mg/kg/h of SB-269970 did not result in any sig-
nificant changes in [11C]Cimbi-806 distribution volumes. Contrary
to the expected displacement, and increased uptake of [11C]Cimbi-
806 was observed in the thalamus, as seen on the TACs (Fig. 2B).
This effect was only observed in the thalamus region and not in
any other region investigated. All metabolite corrected plasma
input functions were analyzed but no significant changes were
observed in input function between baseline and blocked (SB-
269970) conditions. The difference in uptake cannot be ascribed
to blocking of peripheral 5-HT7 receptor sites by the SB-269970
compound.

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M. M. Herth et al. / Bioorg. Med. Chem. 20 (2012) 4574–4581
Br
Br
Br
(a)
Br
NH2
NH2
1. NsCl, Et3N, CH2CL2
0 °C to RT, 1h
2. MeI, NaH, DMF
0 °C to RT, 14h
aq. MeNH2, Cs2CO3
Boc2O, NaHCO3
THF, H2O, Dioxane
RT, 12h,
DMF
reflux, 1h,
12%
96%
98%
Br
Br
Br
NHBoc
NHMe
NMeNos
(2)
(1)
(3)
(b)
MeO
OMe
THF
-78 °C to RT
31%
n-BuLi,
B(OCH3)3
B(OH)2
Br
MeO
OMe
OMe
MeO
+
Pd(PPh3)4, Na2CO3,
toluene, H2O
130 °C, MW, 2-4h
Ar
NR2
NR2
(4)
(1) R = H, Boc
(2) R = H, Me
(3) R = Me, Nos
(5) R = H, Boc
(6) R = H, Me
(7) R = Me, Nos
(c)
1. MeI, NaH, DMF
0 °C to RT, 12h
2. TFA
DBU, R-SH
MeO
OMeMeCN
MeO
OMe
MeO
OMe
4 eq. LiAlH4, THF
0 °C to reflux, 4h
60 °C, 12h
52 %
NHMe
NNos
NHBoc
(5)
(6)
(7)
aq. CH2O, ZnCl2,
Na BH3CN, MeOH
0 °C to RT, 14h
65 %
TFA
79%
aq. CH2O, ZnCl2,
OMe
NaBH3CN, MeOH
MeO
MeO
OMe
0 °C toRT, 14h
82 %
NH2
NMe2
(8)
Cimbi 806
Scheme 1. Chemical syntheses: (a) Synthesis of 3-bromophenethylamine derivatives (b) Synthesis route for the biphenyl core (c) Synthesis of precursor and reference
compound of [11C]Cimbi-806.

M. M. Herth et al. / Bioorg. Med. Chem. 20 (2012) 4574–4581
4577
in rat,22 makes it unlikely that the lack of blockade in this study
is caused by insufficient BBB penetration of the blocking agent.
Thus, since it was not displaced by the selective compound SB-
269970, we could not determine if [11C]Cimbi-806 in vivo binding
is selective for the 5-HT7 receptor. The lack of displacement by SB-
269970 could also be due to the radioligand having non-target
affinity or because of high non-specific binding in the brain.
An autoradiographic study with [3H]SB-269970 to visualize the
distribution of 5-HT7 receptors in human whole hemisphere brain
sections found that binding was mainly found in the thalamus,
hypothalamus and hippocampal formation of the human brain,16
which was in good agreement with autoradiographic studies car-
ried out on rat and guinea-pig brain sections.23,24 However differ-
ences were found when looking at the striatum, where the 5-HT7
selective radioligand [3H]SB-269970 shows low binding and the
less selective radioligand [3H]mesulergine found high binding.25
The autoradiographic experiments carried out on pig brain sections
in this study showed high binding in the cortical areas but also rel-
atively high binding in the striatum (Fig. 1A). With this in mind we
expect high binding of [11C]Cimbi-806 in the thalamus and med-
ium binding in the striatum and cortex. Binding in the cerebellum
is expected, as 5-HT7 receptors have been found in pig cerebel-
lum.21 VT values were indeed higher in thalamus and striatum
and lower in the cerebellum (Fig. 3). Compared to human data, high
striatal binding was not expected but as seen on the autoradio-
graphic images it is likely that a higher density of 5-HT7 receptors
can be found in the pig striatum, and thereby accounting for the
unexpected high uptake of [11C]Cimbi-806.
2.6. In vivo metabolism of [11C]Cimbi-806
After intravenous injection, [11C]Cimbi-806 was rapidly metab-
olized (Fig. 4) and this generated two radiolabelled metabolites
that both were less lipophilic than [11C]Cimbi-806. The parent
compound fraction at baseline and pre-treatment conditions,
showed no significant difference, indicating that pretreatment
with SB-269970 did not affect [11C]Cimbi-806 metabolism. The
free fraction of [11C]Cimbi-806 in plasma (fP) was 23.5 5.0%
(mean SD, n = 10) after 3 h when equilibrium between the dialy-
sis chambers was reached.
Figure 1. (A) In vitro competition autoradiography images with [3H]SB-269970 and
two concentrations of Cimbi-806 (left: 1.56 nM, right: 250 nM). (B) Inhibition of
[3H]SB-269970 binding to pig brain sections by Cimbi-806.
In all pigs, full arterial plasma parent compound input curves
were measured and these were used to calculate the distribution
volumes (VT) of [11C]Cimbi-806 in volumes of interest (VOIs) using
a one-tissue compartment (1-TC) model. An averaged metabolite
curve (based on seven separate injections) was used to correct
plasma activity for parent compound fraction as previously
described20
No significant changes in VT after SB-269970 pre-treatment was
found in any region (P >0.5, 2-way analysis of variance (ANOVA),
Bonferroni post-test) (Fig. 3). High similarity between the deduced
amino acid sequences of human and pig 5-HT7 receptors21 and a
pharmacokinetic study confirming BBB penetration of SB-269970
3. Conclusion
Cimbi-806 and its desmethyl precursor were synthesized in suf-
ficient yields, and 11C-radiolabeling produced [11C]Cimbi-806 in
high specific radioactivity, high radiochemical purity and yield.
Evaluation of [11C]Cimbi-806 in pigs with PET scanning revealed
high brain uptake, and appropriate radiometabolism with only
two polar metabolites, unlikely to cross the BBB, appearing in pig
plasma. Since [11C]Cimbi-806 could not be displaced by the
[
11C]CH3OTf,
Acetone,
60 °C, ~ 100%
MeO
OMe
MeO
OMe
NHMe
NMe
(6)
[11C]Cimbi-806
11CH3
Scheme 2. Radiosynthesis of [11C]Cimbi-806.

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M. M. Herth et al. / Bioorg. Med. Chem. 20 (2012) 4574–4581
Figure 2. (A) Representative PET images summed from 0 to 90 min scanning showing regional uptake of [11C]Cimbi-806 (right column) and the standardized MRI-based atlas
for the pig brain to which the PET images are co-registered (left column). (B) Time-activity curves at baseline (solid lines, n = 5) and blocked (dashed lines, n = 3) conditions.
Bsl = baseline, Tha = thalamus, Str = striatum, Cb = Cerebellum, TACs are represented as standardized uptake value (SUV) S.E.M.
selective 5-HT7 receptor antagonist SB-269970 in vivo, we do not
find this PET radioligand to be suitable for imaging the 5-HT7
receptor.
4. Experimental details
4.1. Chemicals and equipment
Chemicals were purchased from Acros, Fluka, Sigma, Tocris, ABX
or Merck. Unless otherwise stated, all chemicals were used without
further purification. [3H]SB-269970 was purchased from PerkinEl-
mer. Microwave-assisted synthesis was carried out in a Biotage Ini-
tiator apparatus operating in single mode; the microwave cavity
producing controlled irradiation at 2.45 GHz (Biotage AB, Uppsala,
Sweden). The reactions were run in sealed vessels (0.5–2.0 mL).
These experiments were performed by employing magnetic stir-
Figure 3. Distribution volumes (VT) of [11C]Cimbi-806. Mean S.E.M. distribution
volumes for one-tissue compartment (1TC) modeling of baseline (n = 4) and blocked
(n = 3) for the different brain regions.
ring and a fixed hold time using variable power to reach (during
1–2 min) and then maintain the desired temperature in the vessel
for the programmed time period. The temperature was monitored
by an IR sensor focused on a point on the reactor vial glass. The IR
sensor was calibrated to internal solution reaction temperature by
the manufacturer. GC–MS were performed on a Shimadzu. For So-
lid Phase Extraction (SPE), Sep-PakÒ-C18-cartridges (Waters, USA)
were used. Thin Layer Chromatography (TLC) was performed using
plates from Merck (Silicagel 60 F254 and Alumina oxide 60 F254).
1H-NMR spectra were recorded using a Bruker AC 300 spectrome-
ter. Chemical shifts are quoted as d-values (ppm) downfield from
tetramethylsilane (TMS). Field desorption (FD) mass spectra were
recorded using a Finnigan MAT90 spectrometer. Analytical and
preparative high performance liquid chromatography (HPLC) were
performed on a Dionex system consisting of a pump P680A pump,
a UVD 170U detector and a Scansys radiodetector. Metabolite anal-
ysis of pig plasma was performed using a Dionex Ultimate 3000
HPLC system consisting of a DGP-3600SD pump and an online
Posi-Ram Radio Flow-Through Detector. Brain slices were cut on
a HM500OM Cryostat (Microm Intl GmbH). Fuji imaging plates
(IP) were scanned by a Fuji BAS-2500 scanner. PET scanning was
Figure 4. [11C]Cimbi-806 (solid line) and metabolites (dashed lines) as a function of
time after intravenous injection. The graph shows the average of measurements
from seven experiments, error bars represent S.D.

M. M. Herth et al. / Bioorg. Med. Chem. 20 (2012) 4574–4581
4579
performed with a high-resolution research tomography scanner
(HRRT, Siemens AG).
11C]Methane was produced via the
14N(p, 11C reaction by bombardment of an [14N]N2 containing
10% H2 target with a 17 MeV proton beam in a Scanditronix
MC32NI cyclotron.
4.2.5. General procedure for the Suzuki cross-coupling
[
The appropriate phenylbromide (0.517 mmol), 2,6-dimethoxy-
benzeneboronic acid (170 mg, 0.9 mmol) and Na2CO3 (137.65 mg,
1.29 mmol) were dissolved in a mixture of toluene/H2O (10:1)
(5.5 mL) and sparged with argon for at least 30 min to assure all
oxygen is removed from the reaction mixture. Pd(PPh3)4
(29.86 mg, 0.026 mmol) were added and heated for 2–4 h in a
microwave oven (Initiator, Biotage) at 130 °C. The resulting reac-
tion mixture was quenched with water and extracted by diethyl-
ether. After drying with MgSO4 the united organic layers were
reduced and the crude mixture was purified by column chroma-
tography yielding in the desired product.
a)
4.2. Organic synthesis
4.2.1. [2-(3-Bromo-phenyl)-ethyl]-carbamic acid tert-butyl
ester (1)
To a solution of 3-bromophenethylamine (3.12 g, 15.7 mmol)
and NaHCO3 (2.3 g, 27.38 mmol) in THF (50 mL), H2O (50 mL)
and dioxane (50 mL) were added (Boc)2O (4.1 g, 18.8 mmol). This
mixture was stirred for 12 h and then extracted with CH2Cl2. The
combined organic layers were dried, filtered and evaporated to
yield 1 (4.66 g 98%) as a colorless liquid. 1H NMR (300 MHz, CDCl3):
d 7.30–7.27 (m, 2H, ArH), 7.11–7.07 (m, 2H, ArH), 3.30 (q, J = 6 Hz,
2H, NCH2), 2.72 (t, J = 9 Hz, 2H, ArCH2), 1.40 (s, 9H, tert-butyl); TLC
Rf: 0.52 (Heptane/EtOAc 3:1) (full analytical data are reported in26).
4.2.6. tert-Butyl (2-(20,60-dimethoxy-[1,10-biphenyl]-3-yl)ethyl)
carbamate (5)
Colorless liquid (173 mg 94%); 1H NMR (300 MHz, CDCl3): d
7.30–7.12 (m, 5H, ArH), 6.67–7.07 (d, J = 9 Hz 2H, ArH), 3.75 (s,
6H, OCH3), 3.45 (q, J = 6 Hz 2H, NCH2), 2.85 (t, J = 9 Hz 2H, ArCH2),
1.48 (s, 9H, tert-butyl); 13C NMR (75 MHz, CDCl3): d 157.72, 156.04,
138.27, 134.41, 131.61, 129.17, 128.88, 128.15, 127.45, 119.44,
104.39, 79.44, 56.29, 42.00, 35.47, 28.84; TLC Rf: 0.31 (Heptane/
EtOAc 3:1); GC–MS (EI) m/z (% rel Int.): 357 (100.0 [M]+), rt:
16.307 min.
4.2.2. 2-(3-Bromophenyl)-N-methylethanamine (2)
To a solution of 3-bromophenethylbromide (1 g, 3.83 mmol) in
DMF (10 mL) was added 40% aqueous methylamine (2.97 mL,
38.3 mmol), followed by Cs2CO3 (1.625 g, 5 mmol). After the reac-
tion mixture was refluxed for 1 h, it was cooled to room tempera-
ture and the solvent was removed. The residue was dissolved in
ethyl acetate (30 mL), and the organic layer was washed with
water (3 ꢁ 20 mL) and dried over Na2SO4. The solvent was re-
moved in vacuo, and the oily residue was further purified using
flash column chromatography (CHCl3/MeOH 5:2) yielding in 2
(100 mg, 12%) as a colorless liquid. 1H NMR (300 MHz, CDCl3): d
7.26–7.23 (m, 2H, ArH), 7.06–7.04 (m, 2H, ArH), 2.78–2.74 (m,
4H), 2.30 (s, 3H, NCH3); TLC Rf: 0.2 (CHCl3/MeOH 5:2); GC–MS
(EI) m/z (% rel Int.): 214 (100.0 [M]+), rt: 7.240 min (full analytical
data are reported in27).
4.2.7. 2-(20,60-Dimethoxy-[1,10-biphenyl]-3-yl)-N-methyl-
ethanamine (6)
Colorless liquid (116 mg; 83%); 1H NMR (300 MHz, CDCl3): d
7.31–7.14 (m, 5H, ArH), 6.63 (d, J = 6 Hz 2H, ArH), 3.71 (s, 6H,
OCH3), 2.89–2.81 (m, 4H, ArCH2CH2N), 2.43 (s, 3H, NCH3), 1.64
(br s, 1H, NH); TLC Rf: 0.15 (EtOAc/Et3N 3:1); GC–MS: 271 g/mol,
rt: 12.806 min, purity: 100% (full analytical data are reported in29).
4.2.8. N-(2-(20,60-Dimethoxy-[1,10-biphenyl]-3-yl)ethyl)-N-
methyl-2-nitrobenzenesulfonamide (7)
White solide (205 mg; 87%); mp: 128–129 °C; 1H NMR
(300 MHz, CDCl3): d 7.90–7.87 (m, 1H, ArH), 7.63–7.54 (m, 3H,
ArH), 7.28–7.26 (m, 2H, ArH), 7.20–7.17 (m, 2H, ArH), 7.11–7.09
(m, 1H, ArH), 6.63 (d, J = 9 Hz 2H, ArH), 3.71 (s, 6H, OCH3), 3.49
(t, J = 9 Hz, 2H, NCH2), 2.95–2.90 (m, 5H, NCH3 and ArCH2); 13C
NMR (75 MHz, CDCl3): d 157.73, 137.32, 134.66, 133.48, 132.84,
131.74, 131.64, 130.95, 129.48, 128.94, 128.14, 127.41, 125.58,
124.18, 119.33, 104.40, 56.23, 56.13, 35.36, 35.29; TLC Rf: 0.5 (Hep-
tane/EtOAc 1:1).
4.2.3. N-(3-Bromophenethyl)-N-methyl-2-nitrobenzenesulfo-
namide (3)
2-(3-Bromophenyl)ethanamine (2 g, 10.05 mmol) were dis-
solved in anhydrous CH2Cl2 (50 mL) and Et3N (2 g, 20 mmol,
2.77 mL) and cooled to 0 °C. 2-nosylchloride (2.21 g, 10 mmol)
was added dropwise and the mixture was warmed to rt and stirred
for 1 h. The mixture was diluted with Et2O, quenched with 10% aq
HCl and washed successively with sat. NaHCO3 and brine. After
drying with Na2SO4, the solvent was evaporated to yield 3.8 g of
crude product (ꢀ100%) which was used without any further puri-
fication and dissolved in DMF (10 mL). NaH (60% dispersion in
mineral oil, 397.2 mg, 10 mmol) and after 15 min, MeI (0.91 g,
5.28 mmol, 0.4 mL) were added at 0 °C. The mixture was stirred
at room temperature for 14 h, hereafter, DMF was removed. The
resultant mixture was diluted in a mixture of EtOAc/H2O and ex-
tracted with ethyl acetate three times. The combined organic lay-
ers were dried over Na2SO4 and concentrated. The residue was
purified by flash chromatography on silica gel with EtOAc/Heptane
(2:1) yielding in 3 (3.87 g, 96%) as a yellowish liquid. 1H NMR
(300 MHz, CDCl3): d 7.95–7.92 (m, 1H, ArH), 7.70–7.60 (m, 3H,
ArH), 7.37–7.27 (m, 2H, ArH), 7.19–7.11 (m, 2H, ArH), 3.51–3.46
(m, 2H, NCH2), 2.99–2.87 (m, 5H, NCH3 and ArCH2); 13C NMR
4.2.9. 2-(20,60-Dimethoxy-[1,10-biphenyl]-3-yl)-N-methylethana-
mine (6)
A 100 mL round-bottom flask was charged with of 1-dodecyl-
mercaptan (0.722 g, 3.57 mmol, 0.854 mL) and acetonitrile
(25 mL). The mixture is cooled in an ice-water bath and of DBU
(0.542 g, 3.57 mmol, 0.533 mL) was added over
a period of
10 min. After 5 min, the ice-water bath was removed, and 7
(791 mg, 1.785 mmol) in acetonitrile (20 mL) is added and the mix-
ture was heated to 60 °C for 12 h. The crude mixture was taken up
in EtOAc and washed with Na2CO3 and dried over MgSO4, filtered,
and concentrated under reduced pressure. The residue was puri-
fied by column chromatography on silica. First the column was
flushed with (Heptane/EtOAc 1:1) to remove all byproducts and
then with EtOAc/ET3N (2:0.3) to yield the desired secondary amine
as a colorless oil (250 mg, 52%). (See above for analytical data)
(75 MHz, CDCl3):
d 148.15, 140.49, 133.84, 132.47, 132.00,
131.93, 130.80, 130.43, 130.01, 127.79, 124.36, 122.71, 51.81,
35.32, 34.69; TLC Rf: 0.8 (Heptane/EtOAc 2:1); GC–MS (EI) m/z (%
rel Int.): 398.0 (100.0 [M]+), rt: 22.298 min.
4.2.10. 2-(20,60-Dimethoxy-[1,10-biphenyl]-3-yl)ethanamine (8)
Compound 5 (0.105 mg, 0.294 mm) were carefully dissolved in
TFA (5 mL). The resulting mixture was stirred for 4 h. Afterwards,
TFA was removed in vacuo and the resulting mixture solved in
diethylether. Then the pH was set to 8 by addition of 1 M NaHCO3
and then extracted with diethylether (3ꢁ). After drying with
4.2.4. 2,6-Dimethoxybenzeneboronic acid (4)
2,6-Dimethoxybenzeneboronic acid was synthesized as
reported.28

4580
M. M. Herth et al. / Bioorg. Med. Chem. 20 (2012) 4574–4581
Na2SO4 the united organic layers were reduced to yield in the de-
sired product (60 mg, 79%) as a colorless liquid. 1H NMR (300 MHz,
CDCl3): d 7.34–7.10 (m, 5H, ArH), 6.50 (d, J = 6 Hz, 2H, ArH), 4.79
(br s, 2H, NH2), 3.71 (s, 6H, OCH3), 3.04 (t, J = 6 Hz, 2H NCH2),
2.87 (t, J = 6 Hz, 2H, ArCH2); 13C NMR (75 MHz CDCl3): d 157.67,
138.01, 134.50, 131.64, 129.33, 128.93, 128.17, 127.51, 111.34,
104.40, 56.18, 42.89, 38.16; TLC Rf: 0.01 (Heptane/EtOAc 3:1);
GC–MS (EI) m/z (% rel Int.): 257 (100.0 [M]+), rt 12.467 min.
4.4. Lipophilicity
Lipophilicites were determined using a Dionex Ultimate 3000
HPLC equipped with de-gasser, autosampler, column-oven and
UV-detector. The eluent was 50:50 (v/v) 25 mM sodium phos-
pahte-buffer (pH = 7.4) and MeOH. Injected volumes were 100
with a flow rate of 2 mL/min. The column was a Zorbax SB-C8
m). The column oven was kept at 37 °C
lL
(250 mm ꢁ 4.6 mm, 5
l
and detection was at 254 nm. The logarithm of retention factor
of reference compounds (phenol, acetophenone, p-cresol, benzene,
toluene, chlorobenzene, benzophenone, naphthalene, diphenyl and
phenanthrene) and tested compounds was calculated, and a plot of
the reference values against their known logD values was used to
calculate logD values for tested compounds.
4.2.11. General procedure to synthesize 2-(20,60-dimethoxy-
[1,10-biphenyl]-3-yl)-N,N-dimethylethanamine (Cimbi-806)
To a stirred solution of the appropriate amine (1.32 mmol), zinc
chloride (90 mg, 0.65 mmol), and 37% aqueous formaldehyde
(0.4 mL, 5.28 mmol) in MeOH (10 mL) was added sodium cyano-
borohydride (99.64 mg, 1.584 mmol) at 0 °C. The resulting mixture
was stirred at room temperature for 14 h and then quenched by
addition of 1 N NaOH and extracted with ethyl acetate. The com-
bined organic layers were dried over Na2SO4 and concentrated.
The residue was purified by flash chromatography on silica gel
with EtOAc/Et3N (10:0.1) to yield in a colorless oil. Method A:
Starting from 6 resulting in Cimbi-806 (244 mg, 65%). Method B:
Starting from 8 resulting in the desired product (319 mg, 82%).
1H NMR (300 MHz, CDCl3): d 7.36–7.25 (m, 2H, ArH), 7.20–7.18
(m, 3H, ArH), 6.65 (d, J = 6 Hz, 2H, ArH), 3.74 (s, 6H, OCH3), 2.87–
2.81 (m, 2H, NH2), 2.64–2.58 (m, 2H, ArCH2), 2.32 (s, 6H, NCH3);
13C NMR (75 MHz, CDCl3): d 157.99, 139.67, 134.39, 131.67,
129.00, 128.99, 128.09, 127.54, 104.57, 99.38; 61.93, 56.30,
44.61, 34.88; TLC Rf: 0.2 (Heptane/Et3N 10:0.1); GC–MS (EI) m/z
(% rel Int.): 285 (100.0 [M]+), rt: 11.513 min, purity: >99%.
4.5. Animal procedures
Five female Danish Landrace pigs were used in this study (weight
19 kg). After arrival, animals were housed under standard condi-
tions and were allowed to acclimatize for one week before scanning.
To minimize stress, the animals were provided with straw bedding
and environment enrichment, in the form of plastic balls and metal
chains. On the scanning day, pigs were tranquilized by intramuscu-
lar (im) injection of 0.5 mg/kg midazolam. Anesthesia 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, anesthesia was maintained
by intravenous (iv) infusion of 15 mg/kg/h propofol (B. Braun
Melsugen AG). During anesthesia, animals were endotracheally
intubated and ventilated (volume 250 mL, frequency 16 per min).
Venous access was granted through two catheters (Becton Dickin-
son) in the peripheral milk veins, and an arterial line for blood sam-
pling measurement was obtained by a catheter in the femoral artery
after a minor incision. Vital signs including blood pressure and heart
rate were monitored throughout the duration of the PET scanning.
Immediately after scanning, animals were sacrificed by iv injection
of pentobarbital/lidocain. All animal procedures were approved by
the Danish Council for Animal Ethics (Journal No. 2007/561-1320).
4.3. Radiolabeling of [11C]Cimbi-806
11C-methyl trifluoromethanesulfonate ([11C]MeOTf) produced
using a fully automated system was transferred in a stream of he-
lium to a 1.1-mL vial containing the labeling precursor 6 (0.3–
0.4 mg) and acetone (300
lL). The resulting mixture was heated
at 60 °C for 180 s and then purified by HPLC on a Luna 5
l
m
C18(2) 100 Å column (Phenomenex Inc.) (250 ꢁ 10 mm; 25:75 ace-
tonitrile: 0.1% phosphoric acid; and flow rate, 6 mL/min, retention
times: [11C]Cimbi-806 = 7 min; precursor (6) = 5.4 min). The frac-
tion corresponding to the labeled product (11.5 min) was collected
in sterile water (150 mL), and the resulting solution was passed
through a solid-phase C18 Sep-Pak extraction column (Waters
Corp.), which had been preconditioned with ethanol (10 mL), fol-
lowed by isotonic sodium chloride solution (20 mL). The column
was flushed with sterile water (3 mL). Then, the trapped radioactiv-
ity was eluted with ethanol (3 mL), followed by isotonic sodium
chloride solution (3 mL) into a 20-mL vial containing phosphate
buffer (9 mL, 100 mM, pH 7), giving a 15 mL solution of [11C]Cim-
bi-806 with a pH of approximately 7. In a total synthesis time of
80–90 min, 0.6–1.8 GBq of [11C]Cimbi-806 was produced.
4.6. PET scanning protocol
[
11C]Cimbi-806 (n = 7) was given as intravenous bolus injec-
tions, and the pigs were subsequently PET-scanned for 90 min in
list mode with the HRRT scanner. Scanning began at the time of
injection. After the baseline scan, pigs were maintained in anesthe-
sia and scanned a second time using the same PET-protocol. The 5-
HT7 antagonist, SB-269970 was administered 30 min prior to the
second scan (0.5–1.0 mg/kg/h infusion). Infusion was continued
for the remaining of the scan. An average of 468 31 MBq was in-
jected of [11C]Cimbi-806, the average specific activity at the time of
injection was 123 GBq/
l
mol (range: 30–215 GBq/
lmol), and the
average mass injected 1.6
l
g (range: 0.61–4.3 g). During the first
l
4.3.1. Determination of radiochemical purity and specific
radioactivity
30 min of scanning, radioactivity in whole blood was continuously
measured using an ABSS autosampler (Allog Technology) counting
coincidences in a lead-shielded detector. Concurrently, blood sam-
ples were manually drawn at 2.5, 5, 10, 20, 30, 40, 50, 70, and
90 min and the radioactivity in whole blood was measured using
a well counter (Cobra 5003, Packard Instruments) that was cross-
calibrated to the HRRT scanner and autosampler.
The radiotracer preparation was visually inspected for clarity,
and absence of color and particles. Chemical and radiochemical
purities were assessed on the same aliquot by HPLC analysis. Spe-
cific activity (As) of the radiotracers were calculated from three
consecutive HPLC analyses (average) and determined by the area
of the UV absorbance peak corresponding to the radiolabeled prod-
uct on the HPLC chromatogram and compared to a standard curve
4.7. Quantification of PET data
relating mass to UV absorbance (k = 225
lm). Column used for
[
11C]Cimbi-806: Luna 5
l
m C18(2) 100 Å column (Phenomenex
90-minute HRRT list-mode PET data were reconstructed into 38
dynamic frames of increasing length (6 ꢁ 10, 6 ꢁ 20, 4 ꢁ 30,
9 ꢁ 60, 2 ꢁ 180, 8 ꢁ 300, and 3 ꢁ 600 s). Images consisted of 207
planes and 256 ꢁ 256 voxels of 1.22 ꢁ 1.22 ꢁ 1.22 mm. A summed
Inc.) (150 ꢁ 4.6 mm (25:75 acetonitrile: 0.1% phosphoric acid;
and flow rate: 2 mL/min. retention times:
2.618 min; precursor (6) = 2.247 min).
[
11C]Cimbi-806 =

M. M. Herth et al. / Bioorg. Med. Chem. 20 (2012) 4574–4581
4581
picture of all counts in the 90-min scan was reconstructed for each
pig and used for co-registration to a standardized MRI-based atlas
of the Danish Landrace pig brain, similar to that previously pub-
lished.30,31 The temporal radioactivity in volumes of interest
(VOIs), including the cerebellum, cortex, hippocampus, lateral
and medial thalamus, caudate nucleus, and putamen, was ex-
tracted. Activity in the striatum was averaged over the caudate nu-
cleus and putamen. Activity in the thalamus was averaged over the
lateral and medial thalamus. Radioactivity in all VOIs was calcu-
lated as the average of radioactive concentration (Bq/mL) in the left
and right sides. Outcome measure in the time-activity curves was
calculated as radioactive concentration in VOI (in kBq/mL) normal-
ized to the injected dose corrected for animal weight (in kBq/kg),
yielding standardized uptake values (SUV, in the unit of g/mL).
In all pigs, full arterial function was measured. A population-
based averaged metabolite curve of the seven scans was con-
structed and subsequently used to correct plasma activity in the
individual scans for parent compound fraction. We calculated the
distribution values (VT) VOIs for [11C]Cimbi-806 using plasma
corrected for parent compound as arterial input function.
The one-tissue compartment (1TC) model was chosen for quantitative
analysis the data Kinetic modeling was done with PMOD software
(version 3.0; PMOD Technologies Inc.).
of concentration of Cimbi-806 and regressed using Prism 4.0 soft-
ware to obtain Ki.
Acknowledgments
The authors wish to thank the staff at the PET and Cyclotron
unit for expert technical assistance. We also want to thank Mette
Værum Olesen and Letty Klarskov for excellent animal preparation
as well as Lasse Kofoed Bech for logD measurements. Financial
support by Intra European Fellowship (MC-IEF-275329), The Fac-
ulty of Health, University of Copenhagen, and the Lundbeck Foun-
dation is gratefully acknowledged. The John & Birthe Meyer
Foundation and The Toyota foundation are acknowledged for
granting the HRRT scanner and the HPLC system, respectively.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.05.005.
References and notes
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4.8. HPLC analysis of pig plasma
[
11C]Cimbi-806 was separated from its radio-labelled metabo-
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4.9. Protein binding
The free fraction of [11C]Cimbi-806 in plasma, fp, was estimated
using an equilibrium dialysis chamber method as previously de-
scribed.31 Briefly, the dialysis was conducted in chambers (Harvard
Biosciences) separated by cellulose membrane with a protein cut-
off of 10,000 Da. Small amounts of [11C]Cimbi-806 (ꢀ5 MBq) were
added to 5 mL plasma sample from the pig. Plasma (500 lL) was
then dialyzed at 37 °C against an equal volume of buffer
(135 mM NaCl, 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 after various dialysis
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Twenty micrometers coronal sections of pig brain (weight
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and thawed-mounted on super frost plus glass slides, air-dried
and stored at ꢂ80 °C until use. Sections were cut so both cortical
and striatal areas were visible. Autoradiography was performed
with 5 nM [3H]SB-269970 (PerkinElmer, Inc.) and with increasing
competing concentrations of Cimbi-806 (1.56–250 nM). Non-
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28. Fukuda, T.; Sudo, E.-I.; Shimokawa, K.; Iwao, M. Tetrahedron 2008, 64, 328.
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H. H.; Patent number EP1997493; 2008.
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specific binding was determined with 5 lM SB-258719 (Tocris
Bioscience). Two separate experiments were carried out on adjacent
sections from the same pig. Assay buffer used consistend of 50 mM
Tris–HCl, pH 7.4, and an incubation time of 2 h was used. Sections
were washed 3 ꢁ 5 min in ice-cold assay buffer with a subsequent
dip in ice-cold dH2O. Sections were dried and exposed to Fujifilm
tritium-sensitive imaging plates for 14 days. Specific radioligand
binding (total minus non-specific binding) was plotted as a function
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