Synthesis and in vitro/in vivo pharmacological evaluation of [ 11C]-ThioABP, a novel radiotracer for imaging mGluR5 with PET

Article abstract of DOI:10.1039/c2md20332d

We have designed a novel positron emission tomography (PET) radiotracer, [¹¹C]-ThioABP, a thiazole based derivative for imaging the metabotropic glutamate receptor subtype 5 (mGluR5), and prepared the hydroxy oxime precursor 4 in a 15% overall yield. [¹¹C]-ThioABP was radiosynthesized in the Veenstra module and obtained in a decay corrected radiochemical yield of 40% and specific activity of 80-250 GBq μmol ^-1 at the end of synthesis. ThioABP exhibited excellent binding affinity (K_i) in vitro of 1.9 ± 0.9 nM and [ ¹¹C]-ThioABP showed an optimal log D_7.4 of 2.4. The autoradiographic studies on rat brain slices revealed specific binding to mGluR5. In vivo evaluation of [¹¹C]-ThioABP including a displacement study with MMPEP in a dynamic PET scan showed a specificity of [ ¹¹C]-ThioABP for mGluR5. Radio-TLC metabolite studies showed a good metabolic stability of [¹¹C]-ThioABP in vivo. The comparison of biological properties of [¹¹C]-ThioABP and [¹¹C]-ABP688 revealed similarity between these two compounds.

Full text of DOI:10.1039/c2md20332d

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Synthesis and in vitro/in vivo pharmacological
evaluation of [11C]-ThioABP, a novel radiotracer for
imaging mGluR5 with PET†
Cite this: Med. Chem. Commun., 2013,
4, 520
a
b
a
*
¨
Selena Milicevic Sephton, Linjing Mu, Adrienne Muller, Cindy A. Wanger-
Baumann,a Roger Schibli,ab Stefanie D. Kramer and Simon M. Ametamey
a
a
*
¨
We have designed a novel positron emission tomography (PET) radiotracer, [11C]-ThioABP, a thiazole based
derivative for imaging the metabotropic glutamate receptor subtype 5 (mGluR5), and prepared the
hydroxy oxime precursor 4 in a 15% overall yield. [11C]-ThioABP was radiosynthesized in the Veenstra
module and obtained in a decay corrected radiochemical yield of 40% and specific activity of 80–250
GBq mmolꢀ1 at the end of synthesis. ThioABP exhibited excellent binding affinity (Ki) in vitro of 1.9 ꢁ 0.9
nM and [11C]-ThioABP showed an optimal log D7.4 of 2.4. The autoradiographic studies on rat brain
slices revealed specific binding to mGluR5. In vivo evaluation of [11C]-ThioABP including a displacement
study with MMPEP in a dynamic PET scan showed a specificity of [11C]-ThioABP for mGluR5. Radio-TLC
metabolite studies showed a good metabolic stability of [11C]-ThioABP in vivo. The comparison of
Received 5th November 2012
Accepted 19th December 2012
DOI: 10.1039/c2md20332d
biological properties of
compounds.
[ [
11C]-ThioABP and 11C]-ABP688 revealed similarity between these two
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Alzheimer's diseases) as well as the physiological processes
such as learning and memory.7,10,13–23 Carbon-11 has a short
physical half-life of 20 minutes and efforts have been made
towards the development of a uorine-18 based PET radiotracer
with a longer (110 min) physical half-life. Most of the investi-
gated candidates thus far were structurally related to the
ABP688 scaffold. Among the explored derivatives, the Pike
group followed by the Ametamey group utilized the thiazole
functionality and reported on highly potent analogues [18F]-
SP203 (Fig. 1),24–26 and [18F]-FTECMO (Fig. 1),27 respectively,
both of which exhibited deuorination in vivo in rats. Despite
observed deuorination in rats and non-human primates, [18F]-
SP203 was explored in human subjects and showed limited
radiodeuorination.24 [18F]-SP203 has less resemblance to the
Introduction
In recent years, positron emission tomography (PET) has
emerged as a non-invasive molecular imaging technique
capable of facilitating drug development through quantication
of biochemical and pharmacological parameters. The further
advancement of the PET imaging modality is among other
factors dependent on the development of new PET radiotracers
which specically and selectively bind to their biological targets
of interest. PET radiotracers can be any bioactive or synthetic
molecules labelled with positron emitting nuclide (e.g., uo-
rine-18 or carbon-11).1 Most commonly, extensive structure–
activity relationship (SAR) studies are required for identication
of new PET radiotracers and one such successful example is an
SAR study for the establishment of [11C]-ABP688 (Fig. 1).2–4 To
date, [11C]-ABP688 is the most successful clinically applied PET
radiotracer for imaging metabotropic glutamate receptor
subtype 5 (mGluR5) in human subjects.5–12 Our particular
interest in mGluR5 arose from its involvement in several central
nervous system (CNS) diseases (e.g., Parkinson's and
ABP688 manifold and exhibited superior binding affinity (IC50
)
of 36 ꢁ 9 pM. On the other hand, the structure of [18F]-FTECMO
differed from the structure of ABP688 in that the pyridine
moiety from ABP688 was replaced by the thiazole functionality
and showed a Ki value of 5.5 ꢁ 1.1 nM. With the goal of further
probing the thiazole functionality on the [11C]-ABP688 scaffold
and to ascertain whether a new ligand with improved in vivo
binding properties could be generated, we sought to explore a
new carbon-11 labelled ABP688 analogue, [11C]-ThioABP. In
effect, combining the ABP688 cyclohexenyl subunit labelled
with carbon-11 with the more active thiazole component
allowed for a new structural construct which could be a
potentially useful mGluR5 imaging agent. We herein report the
synthesis of ThioABP, the radiolabelling and pharmacological
evaluation of [11C]-ThioABP in vitro and in vivo as well as the
aCentre for Radiopharmaceutical Sciences of ETH, PSI and USZ, Department of
Chemistry and Applied Biosciences, ETH Zurich, Wolfgang-Pauli strasse 10, 8093
Zurich, Switzerland. E-mail: simon.ametamey@pharma.ethz.ch; Fax: +41 44
6331367; Tel: +41 44 6337463
bCentre for Radiopharmaceutical Sciences of ETH, PSI and USZ, Department of
¨
Nuclear Medicine, University Hospital Zurich, Ramistrasse 100, 8091 Zurich,
Switzerland. E-mail: linjing.mu@usz.ch; Fax: +41 44 6331367; Tel: +41 44 2559687
† Electronic supplementary information (ESI) available: NMR data for 5, IC50
binding curves, HPLC traces are provided. See DOI: 10.1039/c2md20332d
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Fig. 1 Structures of [11C]-ABP688 and thioazole containing PET radiotracers for
imaging mGluR5.
Fig.
2
[3H]-ABP688 displacement curve for ThioABP. Three independent
experiments were performed. The averaged values and their standard deviations
are represented by symbols. Data were analysed by non-linear regression
assuming one binding site (solid line curve). Fitted IC50 was 3.82 ꢁ 1.55 nM
(indicated by the dotted line).
comparative analysis of the in vivo PET scans of the two radio-
tracers [11C]-ThioABP and [11C]-ABP688.
Results and discussion
Syntheses of ThioABP and [11C]-ThioABP
ꢂ
in DMF at 90 C (Scheme 1). Semi-preparative HPLC was used
for the purication and [11C]-ThioABP was formulated in 5%
ethanol for subsequent in vitro and in vivo characterization. Co-
injection with reference compound 5 conrmed the identity of
the radiolabelled product [11C]-5 ([11C]-ThioABP). In a typical
experiment, the decay corrected radiochemical yield was
around 40% and the specic activity of [11C]-ThioABP was in the
range of 80–250 GBq mmolꢀ1 at the end of synthesis with >99%
radiochemical purity. Notably, [11C]-ThioABP was obtained as a
mixture with a by-product, which was identied as a Z-isomer.
The E : Z ratio of the formulated product [11C]-ThioABP was
>20 : 1 similar to that of [11C]-ABP688.2,30
ThioABP was synthesized analogously to the synthesis of
ABP688 and the synthetic scheme is depicted in Scheme 1.
Bromomethyl thiazole 2 was obtained by lithiation of the
commercially available dibromothiazole 1 with n-butyllithium
and concomitantly reacted with dimethyl sulfate in 38% yield
(Scheme 1).28 Sonogashira reaction of thiazole 2 with cyclo-
hexenyl oxime 3 afforded hydroxy oxime 4 in 41%, which was
used as a precursor for the radiolabelling. The corresponding
standard reference, methyl oxime ether 5 (ThioABP) was
synthesized from oxime 4 and methyliodide in a 32% yield. All
compounds were characterized by proton and carbon NMR
spectroscopy, IR and MS. The assignment of the oxime ether
In vitro and in vivo evaluation of [11C]-ThioABP
double bond conguration in
3 has been previously
established.29
The lipophilicities of [11C]-ABP688 and [11C]-ThioABP were
expected to be similar based on their calculated (clogP) values
(2.4 and 2.3, respectively). The distribution coefficient log D7.4
of [11C]-ThioABP in octanol–phosphate buffer was 2.4 ꢁ 0.1
(n ¼ 4), identical to the log D7.4 value reported for [11C]-ABP688
(2.4 ꢁ 0.1).2
Radiolabelling of precursor 4 was performed in an auto-
mated Veenstra module. [11C]-CO2 was produced in the cyclo-
tron via the 14N(p,a)11C nuclear reaction using a nitrogen gas
target in the presence of 0.5% of oxygen. [11C]-CO2 was then
reduced with hydrogen over RANEYꢀ nickel to [11C]-CH4
followed by the reaction with I2 at high temperature to yield
[11C]-CH3I. Reaction with I2 is repeated in the circulation mode
and is the rate determining step for the carbon-11 labelling.
Finally, [11C]-ThioABP was radiosynthesized by bubbling of
[11C]-CH3I through the reaction mixture of precursor 4 and NaH
Fig. 3 In vitro autoradiography on rat brain slices under baseline and blocking
conditions. Left: high radioactivity accumulation is observed in the brain regions
with highest mGluR5 expression under baseline conditions (1 nM [11C]-ThioABP);
middle: complete blocking is achieved by co-incubation with cold ABP688 (100
nM); right: [11C]-ThioABP shows selectivity for mGluR5 over mGluR1 as confirmed
by co-incubation with excess mGluR1 antagonist JNJ16259685. Note: Slices used
for the experiment do not contain striatal brain regions. The slides correspond to
the plates designated as PW116, bregma ꢀ3.10 mm in Paxinos and Watson, The
Rat Brain Atlas, 1998 edition.
Scheme 1 Synthesis and radiolabelling of precursor 4 and the preparation of
cold reference 5.
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value using the Cheng–Prusoff equation (see the Experimental
section). Three independent experiments performed in tripli-
cate revealed a favourable Ki of 1.9 ꢁ 0.9 nM which is within the
range required for an optimal mGluR5 PET tracer for imaging in
the brain and comparable to that of ABP688 (2.4 nM).2 The
displacement curve showing the averaged values of three IC50
measurements is depicted in Fig. 2.
The high binding affinity of ThioABP measured in the
displacement assay prompted us to further evaluate whether
binding of [11C]-ThioABP was selective and specic for mGluR5.
For this purpose, an in vitro autoradiography was performed on
rat brain slices with [11C]-ThioABP under baseline (tracer only)
and blocking (ABP688 or JNJ16259685) conditions (Fig. 3).
Incubation of slices with 1 nM solution of [11C]-ThioABP
resulted in a heterogeneous distribution of radioactivity with
the highest accumulation in the regions where mGluR5 is
highly expressed (e.g., hippocampus and cortex) and back-
ground levels in cerebellum where mGluR5 expression is
low.21,31 Co-incubation of the brain slices with excess of ABP688
(100 nM) and 1 nM [11C]-ThioABP led to a signicant reduction
Fig. 4
[
11C]-ThioABP and [11C]-ABP688 PET images of a rat (head). Images are
averaged from 2 to 45 min after i.v. injection of 40.5 and 31.2 MBq of [11C]-
ThioABP and [11C]-ABP688, respectively. Both tracers show the highest uptake in
striatum, a region rich in mGluR5.
The binding affinity (Ki) of ThioABP towards mGluR5 was of radioactivity accumulation and a homogenous distribution,
determined via a displacement assay with rat brain membranes indicating saturation of available binding sites by excess
using [3H]-ABP688. The Ki value was estimated from the IC50 ABP688 and therefore specicity of [11C]-ThioABP for mGluR5.
Fig. 5 Time–activity curves (TACs) for [11C]-ThioABPand [11C]-ABP688. (a): TACs for the in vivo dynamic PETscan with [11C]-ThioABP in rat, baseline heterogeneous activity
distribution; (b): TACs for the in vivo dynamic PET displacement scan with [11C]-ThioABP, 1 mg kgꢀ1 MMPEP 30 min p.i., reduction in activity uptake as a result of
displacement with mGluR5 antagonist; (c): direct comparison of TACs for [11C]-ThioABPand [11C]-ABP688 in the brain regions of highest (striatum) and lowest (cerebellum)
expression of mGluR5; (d): [11C]-ThioABP and [11C]-ABP688 ratios between specifically bound and free or non-specifically accumulated tracers in striatum and cerebellum.
522 | Med. Chem. Commun., 2013, 4, 520–526
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On the other hand, application of 1 nM [11C]-ThioABP together
with an excess of mGluR1 antagonist, JNJ16259685 (100 nM),
showed the same radioactivity distribution as under baseline
conditions.
Conclusions
The structure of ThioABP was a combination of the thiazole
component from highly potent uorine-18 labelled mGluR5 PET
tracers which underwent deuorination in vivo and cyclohexenyl
carbon-11 labelled oxime bearing subunit from a clinically
applied carbon-11 based mGluR5 PET tracer [11C]-ABP688.
Evaluation of [11C]-ThioABP in vivo in a dynamic PET scan
showed the highest activity uptake in striatum where mGluR5 is
highly expressed. The specicity and reversibility of [11C]-Thio-
ABP binding to mGluR5 was also conrmed by the displacement
study with the mGluR5 antagonist, MMPEP. In comparison to
[11C]-ABP688, an mGluR5 PET radiotracer applied clinically with
success, [11C]-ThioABP exhibited similar in vitro and in vivo
proles; however, [11C]-ThioABP exhibited a slightly reduced
ratio between the specically bound and free/non-specically
accumulated tracer. The metabolic stability of [11C]-ThioABP was
comparable to that of [11C]-ABP688. Overall, the similarity
observed inthe in vivo behaviour of the twotracers suggestedthat
the introduction of the thiazole functionality did not improve the
in vivo properties of the new thiazole based ligand.
We next evaluated properties of [11C]-ThioABP in vivo. Fig. 4
shows PET/CT images of rat head regions aer [11C]-ThioABP
and [11C]-ABP688 injections, respectively. [11C]-ThioABP shows
the same heterogeneous distribution pattern as [11C]-ABP688
with highest uptake in brain regions with high mGluR5
expression (striatum, hippocampus and cortex) and low
radioactivity in the cerebellum. This distribution pattern is
in agreement with the results of an autoradiographic study
in vitro.
The normalized time–activity curves (TACs) of [11C]-Thio-
ABP, obtained from the PET images, in mGluR5-rich brain
regions and the cerebellum are shown in Fig. 5a. A displace-
ment experiment was also conducted to investigate the speci-
city and reversibility of [11C]-ThioABP binding to mGluR5. To
this end, 1 mg kgꢀ1 of mGluR5 antagonist MMPEP was injec-
ted i.v. 30 min aer tracer application. As seen in Fig. 5b the
application of MMPEP resulted in a reduction of radioactivity
in mGluR5-rich regions to the level of radioactivity typically
observed in the cerebellum, thus suggesting selective and
reversible binding of [11C]-ThioABP to mGluR5. The TACs of
[11C]-ThioABP and [11C]-ABP688 were then compared to show a
Experimental
General techniques
similar overall shape. Normalized radioactivity values for [11C]- All reactions requiring anhydrous conditions were conducted in
ThioABP were higher than those for [11C]-ABP688 in the brain ame-dried glass apparatus under an atmosphere of inert gas.
regions examined. For clarity, only the striatum and cere- All chemicals and anhydrous solvents were purchased from
bellum are depicted in Fig. 5c. Fig. 5d depicts the respective Aldrich or ABCR and used as received unless otherwise noted.
ratios ((activity in striatum – activity in cerebellum)/activity in Compounds 2,28 3,29 and 4 (ref. 27) were prepared as previously
cerebellum) for the two radiotracers. At distribution equilib- described. The reported density values are for ambient
rium, this ratio becomes a constant for a radioligand that temperature. [3H]-ABP688 (2.405 GBq mmolꢀ1, 37 MBq mLꢀ1
binds reversibly to its target and is taken as an estimate of the solution in EtOH) was obtained from AstraZeneca. Preparative
binding potential (BP), i.e., the ratio between a specically chromatographic separations were performed on Aldrich
bound tracer and unbound or non-specically accumulated Science silica gel 60 (35–75 mm) and reactions followed by TLC
tracer.32,33 BP is dependent on the receptor density (Bmax), analysis using Sigma-Aldrich silica gel 60 plates (2–25 mm) with
receptor binding affinity (Kd) and non-specic tissue accumu- a uorescent indicator (254 nm) and visualized with UV or
lation. Comparing the two scans, the ratio of the specic to potassium permanganate. Infrared spectra were recorded on a
free/non-specically accumulated tracer was higher for [11C]- JASCO FT/IR 6200 (OmniLab) spectrometer using a chloroform
ABP688 than for [11C]-ThioABP (considering the data between solution of the compound. 1H and 13C NMR spectra were
10 and 60 min p.i.). BP could not be estimated, because the recorded in Fourier transform mode at the eld strength spec-
equilibration between the specically bound and free/non- ied on Bruker Avance FT-NMR spectrometers. Spectra were
specically bound tracer was not reached within the scan obtained from the specied deuterated solvents in 5 mm
duration of 60 min for either tracer.
diameter tubes. Chemical shi in ppm is quoted relative to
Blood–brain barrier permeating radioactive metabolites residual solvent signals calibrated as follows: CDCl3 dH (CHCl3)
generated by liver metabolism could contribute to background ¼ 7.26 ppm, dC ¼ 77.2 ppm. Multiplicities in the 1H NMR
radioactivity in the brain. Blood samples drawn 10 min aer spectra are described as: s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼
tracer injection showed 37% of parent [11C]-ThioABP and 63% quartet, quint. ¼ quintet, m ¼ multiplet, b ¼ broad; coupling
of other more polar radio-metabolites. More importantly, 90% constants are reported in Hz. Numbers in parentheses following
of intact parent radiotracer was detected in the brain sample at carbon atom chemical shis refer to the number of attached
10 min p.i. These results are comparable to the results obtained hydrogen atoms as revealed by the DEPT spectral editing tech-
for [11C]-ABP688, where 95% of brain radioactivity originated nique. Electrospray (ES) mass spectra (LRMS) were obtained
from the parent radiotracer 30 min aer the [11C]-ABP688 with a Micromass Quattro micro API LC and electrospray ioni-
application.2 In the rat brain homogenates,
[
18F]-SP203 zation and electrospray (ES) mass spectra (HRMS) were
demonstrated signicant radiodeuorination in vivo.25 There- obtained with a Bruker FTMS 4.7 T BioAPEXII spectrometer. Ion
fore, [11C]-ThioABP, analogous to [11C]-ABP688 showed superior mass/charge (m/z) ratios are reported as values in atomic mass
metabolic stability in comparison to [18F]-SP203.
units. Semi-preparative purication of radiolabelled material
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was performed on a Merck-Hitachi L2130 system equipped with nuclear reaction 14N(p,a)11C as follows: the [11C]-CO2 formed
Elite LaChrom HITACHI L-2400 variable wavelength detector was reduced to [11C]-CH4 by reduction using a RANEYꢀ nickel
and a VEENSTRA VRM-202 radiation detector using a reverse catalyst and [11C]-CH4 was reacted with I2 at 720 ꢂC to yield [11C]-
phase column (C8 Waters Symmetry, 5 mm, 7.8 ꢃ 50 mm) and CH3I. [11C]-Methyliodide was then bubbled through the heated
ꢂ
eluting with 0.1% aq. H3PO4 (solvent A) and acetonitrile (solvent (temperature of the reaction 90 C) yellow mixture of (E)-3-((2-
B) as solvents and a gradient from 0 to 5 min 70% B; 5–10 min methylthiazol-4-yl)ethynyl)cyclohex-2-enone oxime (1.08 mg,
30–50% B; 10–20 min 50% B at a ow rate of 4 mL minꢀ1
.
4.65 mmol) in anhydrous N,N0-dimethylformamide (0.2 mL) and
Analytical HPLC samples were analyzed by Agilent HPLC 1100 sodium hydride (0.4 mL, c ¼ 1.7 mg mLꢀ1) in anhydrous N,N0-
system equipped with a UV multi-wavelength detector and dimethylformamide and the mixture was stirred for 3 min aer
Raytest Gabi star radiation detector using a reverse phase which time it was diluted with 30% MeCN in 0.1% aq. H3PO4
column (ACE 111–0546, C18, 3 mm, 50 ꢃ 4.6 mm) and eluting (1.4 mL) and puried by HPLC semi-preparative chromatog-
with 45% aq. MeCN at a ow rate of 1 mL minꢀ1. Further raphy. The collected product was diluted with H2O (12 mL) and
information on a Veenstra automated module for the carbon-11 then passed through pre-conditioned (with EtOH 1 ꢃ 5 mL;
radio-labelling can be found online at: http://www.de. then H2O 1 ꢃ 5 mL) C18 light cartridge and the title compound
veenstranet.com.
was eluted with EtOH (0.5 mL) and the product was formulated
by diluting with 9.5 mL of PEG : H2O (1 : 1) solution. Specic
activity was determined to be 80–250 GBq mmolꢀ1 at the end of
the synthesis and purity >99% in E : Z ratio higher than 20 : 1.
However, the E : Z ratio of [11C]-ThioABP varied and in some
instances pure E-isomer was obtained. Variation was due to the
peak overlap of E- and Z-isomers in semi-preparative HPLC. The
total synthesis time from the end of bombardment was 35 min.
Animals
Male Wistar rats were obtained from Charles River (Sulzfeld,
Germany). Animal care and all experimental procedures were
approved by the Cantonal Veterinary Office in Zurich, Switzer-
land. The animals were allowed free access to food and water.
(E)-3-((6-Methylpyridin-2-yl)ethynyl)cyclohex-2-enone
O-
Chemistry
[
11C]-methyl oxime ([11C]-ABP688). Radiolabelling was per-
(E)-3-((2-Methylthiazol-4-yl)ethynyl)cyclohex-2-enone O-methyl
oxime (5). A ame dried round bottom ask was at ambient
temperature under an atmosphere of N2 charged with (E)-3-((2-
methylthiazol-4-yl)ethynyl)cyclohex-2-enone oxime (70 mg,
0.30 mmol) and anhydrous N,N0-dimethylformamide (3 mL) was
added and the clear yellow solution was then treated with
sodium hydride 60% suspension in oil (14 mg, 0.36 mmol) in
one portion and the resulting dark yellow mixture was stirred
for 40 min. Aer this time the mixture was further treated with a
solution of methyliodide (20 mL, 47 mg, 0.33 mmol, d ¼ 2.28) in
anhydrous N,N0-dimethylformamide (2 mL) dropwise over 4
min and the brown mixture was stirred for 24 h. Aer this time
the mixture was quenched with saturated aq. NaHCO3 (5 mL)
and it was diluted with H2O (5 mL) and EtOAc (10 mL) and the
two layers were well shaken and separated. The aqueous phase
was extracted with EtOAc (2 ꢃ 10 mL). The combined organic
extracts were washed with H2O (3 ꢃ 8 mL), brine (1 ꢃ 9 mL),
dried (Na2SO4) and concentrated in vacuo to give the crude
material as a brown oily residue (727 mg). The crude mixture
was puried by chromatography on a silica gel column (eluting
with 20% EtOAc–pentane) to afford the title compound (18.2
mg, 0.07 mmol, 32%) as a pale yellow solid: IR (CDCl3) 2934,
1497, 1437, 1294, 1170, 1047, 919, 885, 868, 747 cmꢀ1; 1H NMR
(400 MHz, CDCl3) d 7.31 (s, 1H), 6.51 (s, 1H), 3.91 (s, 3H), 2.72 (s,
3H), 2.53 (m, 2H), 2.37 (m, 2H), 1.79 (quint, J ¼ 6.1 Hz, 2H) ppm;
13C NMR (100 MHz, CDCl3) d 165.9 (0), 155.6 (0), 136.9 (0), 130.3
(1), 127.5 (0), 122.7 (1), 89.8 (0), 87.0 (0), 62.1 (3), 29.6 (2), 22.3
(2), 21.0 (2), 19.4 (3) ppm; MS (ES+) m/z 247 (M + H)+; HRMS
(ESI) m/z 247.0899 (calcd for C13H15N2OS: 247.0900).
formed using the same module system as for the synthesis of
[11C]-5 and as previously described.2,30 Specic activity was
determined to be >100 GBq mmolꢀ1 at the end of the synthesis
and purity >99% in an E : Z ratio higher than 10 : 1.
Pharmacological evaluation
Binding affinity. Brain membranes were prepared from
Sprague Dawley rat brains as described previously.29,34 Frozen
membranes were thawed on ice and pelleted at 45 000ꢃ g at 4
ꢂC for 5 min. The membranes were washed twice with HEPES
buffer (30 mM HEPES, 110 mM NaCl, 5 mM KCl, 2.5 mM CaCl2,
1.2 mM MgCl2, pH 8 at 4 ꢂC) and resuspended in HEPES buffer
at a protein concentration of 1.3 mg mLꢀ1. The binding assay
was performed as previously described.29 In brief, brain
membranes (0.1 mg protein) were incubated in triplicates at
ambient temperature with 2 nM [3H]ABP688 and ThioABP at
concentrations between 10 pM and 100 mM in a total volume of
0.2 mL HEPES. ThioABP was diluted from a 1 mM ethanolic
(50%) solution. The corresponding EtOH concentrations did
not affect [3H]ABP688 binding (data not shown). Nonspecic
binding of [3H]ABP688 was estimated with 100 mM MMPEP.
Aer 45 min, the samples were ltered and the lters contain-
ing the membranes with bound [3H]ABP688 were measured in
a b-counter (Beckman LS6500). Bound [3H]ABP688 (B, pmol
per mg protein) was tted with Excel solver to eqn (1) to
estimate IC50.
B ¼ Bmin + ((Bmax ꢀ Bmin)/(1 + (C/IC50)))
(1)
where C is the total ThioABP concentration, Bmax is tted
maximal B, i.e., the plateau in the B/log C plot at low log C and
Radiolabelling
(E)-3-((2-Methylthiazol-4-yl)ethynyl)cyclohex-2-enone O-11C- Bmin is tted minimal B, i.e., the plateau at high log C. The
methyl oxime ([11C]-5). [11C]-Methyliodide was produced via the inhibition constant Ki of ThioABP was estimated from IC50 and
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Kd of ABP688 (1.7 ꢁ 0.2 nM) (ref. 2) with the Cheng–Prusoff
equation.
Acknowledgements
Mr Bruno Mancosu is acknowledged for technical support with
the carbon-11 module. Ms Claudia Keller is acknowledged for
technical support in the PET lab. Dr Mark A. Sephton (ZHAW) is
acknowledged for proofreading the manuscript. Prof. P. A.
Schubiger is acknowledged for useful discussions.
Determination of log D. The determination of log D value
was performed using the shake-ask method as previously
reported.29,35 A formulated solution of [11C]-ThioABP (ca. 4 MBq)
was partitioned between phosphate buffer (pH 7.4) saturated
with 1-octanol (500 mL) and 1-octanol saturated with phosphate
(pH 7.4) buffer (750 mL). The washed octanol phase was divided
into four individual aliquots (150 mL) and each was diluted with
phosphate (pH 7.4) buffer saturated with 1-octanol (150 mL) and
the two phases were shaken and radioactivity in each phase was
measured in a g-counter.
Notes and references
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In vitro autoradiography. Frozen horizontal brain slices
(20 mm) from a male Wistar rat (535 g) adsorbed to SuperFrost
Plus slides were thawed at ambient temperature and pre-
incubated on ice for 10 min in HEPES buffer (see above)
containing 0.1% bovine serum albumin (BSA). Excess solution
was carefully removed and slides were incubated with 1 nM
[11C]-ThioABP alone or together with 100 mM ABP688 or 100
mM JNJ16259685 in HEPES buffer for 45 min at ambient
temperature. Aer incubation, the solutions were decanted
and the slides washed in ice cold HEPES buffer containing
0.1% BSA, and twice in HEPES buffer (3 minutes each) and
nally dipped in H2O. Dried slides were exposed to a phos-
phor imager plate for 30 min and the plate was scanned in a
BAS5000 reader (Fuji).
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In vivo PET/CT scans. Rats were immobilized by anaesthesia
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