Syntheses and pharmacological characterization of novel thiazole derivatives as potential mGluR5 PET ligands

Article abstract of DOI:10.1016/j.bmc.2010.06.070

Four novel thiazole containing ABP688 derivatives were synthesized and evaluated for their binding affinity towards the metabotropic glutamate receptor subtype 5 (mGluR5). (E)-3-((2-(Fluoromethyl)thiazol-4-yl)ethynyl)cyclohex-2- enone O-methyl oxime (FTECMO), the ligand with the highest binding affinity (K_i = 5.5 ± 1.1 nM), was labeled with fluorine-18. [ ¹⁸F]-FTECMO displayed optimal lipophilicity (log D_pH7.4 = 1.6 ± 0.2) and high stability in rat and human plasma as well as sufficient stability in rat liver microsomes. In vitro autoradiography with [¹⁸F]-FTECMO revealed a heterogeneous and displaceable binding in mGluR5-rich brain regions. PET imaging with [¹⁸F]-FTECMO in Wistar rats, however, showed low brain uptake. Uptake of radioactivity into the skull was observed suggesting in vivo defluorination. Thus, although [ ¹⁸F]-FTECMO is an excellent ligand for the detection of mGluR5 in vitro, its in vivo characteristics are not optimal for the imaging of mGluR5 in rats in vivo.

Full text of DOI:10.1016/j.bmc.2010.06.070

Bioorganic & Medicinal Chemistry 18 (2010) 6044–6054
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Syntheses and pharmacological characterization of novel thiazole derivatives
as potential mGluR5 PET ligands
Cindy A. Baumann, Linjing Mu, Nicole Wertli, Stefanie D. Krämer, Michael Honer, Pius A. Schubiger,
*
Simon M. Ametamey
Center for Radiopharmaceutical Sciences, ETH Zurich (Swiss Federal Institute of Technology), Wolfgang-Pauli Strasse 10, 8093 Zurich, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 January 2010
Revised 8 June 2010
Accepted 20 June 2010
Available online 25 June 2010
Four novel thiazole containing ABP688 derivatives were synthesized and evaluated for their binding
affinity towards the metabotropic glutamate receptor subtype 5 (mGluR5). (E)-3-((2-(Fluoromethyl)thia-
zol-4-yl)ethynyl)cyclohex-2-enone O-methyl oxime (FTECMO), the ligand with the highest binding affin-
ity (Ki = 5.5 1.1 nM), was labeled with fluorine-18. [18F]-FTECMO displayed optimal lipophilicity
(log DpH7.4 = 1.6 0.2) and high stability in rat and human plasma as well as sufficient stability in rat liver
microsomes. In vitro autoradiography with [18F]-FTECMO revealed a heterogeneous and displaceable
binding in mGluR5-rich brain regions. PET imaging with [18F]-FTECMO in Wistar rats, however, showed
low brain uptake. Uptake of radioactivity into the skull was observed suggesting in vivo defluorination.
Thus, although [18F]-FTECMO is an excellent ligand for the detection of mGluR5 in vitro, its in vivo char-
acteristics are not optimal for the imaging of mGluR5 in rats in vivo.
Keywords:
Metabotropic glutamate receptor subtype 5
(mGluR5)
PET imaging
Fluorine-18
Ó 2010 Elsevier Ltd. All rights reserved.
Neurological disorders
1. Introduction
disease6,7 or other disorders such as depression,8 anxiety,9 schizo-
phrenia,10,11 neuropathic pain,12,13 drug addiction,14 and fragile X
Glutamate is the predominant excitatory neurotransmitter in
the mammalian central nervous system (CNS). Glutamate recep-
tors form a large family, which can be classified into ionotropic
and metabotropic glutamate receptors. The ligand-gated, cation-
selective ion channels that form the ionotropic glutamate receptors
(iGluRs) mediate fast excitatory neurotransmission. IGluRs include
syndrome.15 However, the function of mGluR5 is not yet well
understood and it is generally agreed that a better understanding
of the physiological and pathophysiological roles of the receptor
will open new avenues for the development of diagnostic tools
and effective drugs for the above mentioned CNS disorders.16
Positron emission tomography (PET) is a non-invasive in vivo
imaging technique that offers the possibility to visualize and ana-
lyze mGluR5 expression under various physiological and patho-
physiological conditions. Our group reported the first successful
in vivo imaging of mGluR5 in rodents and humans using carbon-
11 labeled ABP688 (Fig. 1, 4).17 The short physical half-life of car-
bon-11 (t1/2 = 20 min), however, does not permit the widespread
use of [11C]-ABP688. More advantageous seems the use of fluo-
rine-18 (t1/2 = 110 min) due to the possibility of satellite distribu-
tion of potential fluorine-18 labeled compounds to centers
without a cyclotron facility. Recently, a number of fluorine-18 la-
beled compounds for imaging mGluR5 have been reported. Among
these are [18F]F-PEB18,19 (Fig. 1, 1) and the thiazole derivatives
kainate,
a-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid
(AMPA) and N-methyl-D-aspartate (NMDA) receptors. Metabotro-
pic glutamate receptors (mGluRs) are known to be involved in
the modulation of iGluRs and appear to fine-tune neuronal activity.
The subclass of mGluRs consists of eight G-protein coupled recep-
tors (GPCRs) that are sub-divided according to their receptor phar-
macology, amino acid sequences and their secondary messenger
systems into three groups (groups I–III). Group I comprises
mGluR1 and mGluR5 that are mainly post-synaptic receptors acti-
vating Gq proteins and phospholipase C as a secondary messenger.
Group II consists of mGluR2 and mGluR3 while mGluR4, mGluR6,
mGluR7, and mGluR8 form group III. Receptors of both groups
use a Gi protein for signal transduction.1,2
[
18F]F-MTEB18 (Fig. 1, 2) and [18F]SP20320 (Fig. 1, 3). Microwave
heating was applied for the radiosynthesis of [18F]F-PEB and
18F]F-MTEB but only low radiochemical yields were obtained.
The low radiochemical yields were improved later on when ther-
mal heating was employed.21 18F]F-PEB18,19 was recently used
for the in vivo imaging of mGluR5 in human studies.22 For
18F]SP203 (Fig. 1, 3), radiodefluorination was observed in PET
studies involving monkeys. In humans, however, low uptake of
MGluRs have been implicated in numerous CNS disorders.
Notably, mGluR5, which is predominantly located in the hippo-
campus, striatum, and cortex,3 was shown to be involved in neuro-
degenerative diseases such as Alzheimer’s disease,4,5 Parkinson’s
[
[
[
* Corresponding author.
E-mail address: simon.ametamey@pharma.ethz.ch (S.M. Ametamey).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.070

C. A. Baumann et al. / Bioorg. Med. Chem. 18 (2010) 6044–6054
6045
Figure 1. Structures of mGluR5 radioligands.
radioactivity into the skull was observed, suggesting a lower radi-
odefluorination rate in humans.23 Recently, our group also re-
ported on two novel fluorine-18 labeled analogues of ABP688:
[
[
18F]-FPECMO24 (Fig. 1, 5) and [18F]-FE-DABP68825 (Fig. 1, 6). While
18F]-FPECMO underwent radiodefluorination in vivo in rats, [18F]-
FE-DABP688 displayed unfavorable pharmacokinetics. As part of
our program to develop fluorine-18 labeled derivatives of
ABP688, we designed four novel compounds based on the struc-
tural elements of the two most successful mGluR5 PET ligands,
Scheme 2. Synthesis of model compound 14. Reagents and conditions: (a)
Pd(PPh3)4, CuI, Et3N, DMF, rt, 48 h, 26%.
Sonogashira coupling of the trans-oxime 17,26 to either 2-methyl-
4-bromothiazole or 4-bromothiazole gave acceptable yields of com-
pounds 18 and 19. Both intermediates were reacted with (2-(2-
bromoethoxy)ethoxy)(tert-butyl)dimethylsilane to afford the TBS
protected intermediates 20 and 21, respectively. Compounds 20
and 21 were desilylated using TBAF and reacted with benzylsulfo-
nylchloride to give tosylates 22 and 23. Nucleophilic substitution
reaction of the tosyl leaving group with dry TBAF afforded final prod-
ucts 24 and 25 (Scheme 3).
The precursor for the preparation of the radiolabeled analogue
of compound 11 was synthesized in analogy to the method de-
scribed for the synthesis of the precursor for [18F]SP203.20 Com-
pound 26 was prepared according to the procedure reported by
Siméon et al.20 The Sonogashira coupling of compound 26 and
intermediate 13 was accomplished in reasonable yields and gave
compound 27. Compound 27 was treated with TBAF to yield alco-
hol 28. In the final step, alcohol 28 was converted to the bromide
precursor 29 using CBr4 and PPh3 (Scheme 4).
[
11C]-ABP688 and [18F]SP203. Herein, we report the syntheses
and binding affinities of the four novel thiazole containing
ABP688 derivatives. Furthermore, we report on the radiolabeling,
in vitro and in vivo evaluation of the most promising candidate,
[
18F]-FTECMO.
2. Results
2.1. Chemistry
The syntheses of four novel mGluR5 ligands (11, 14, 24, and 25)
containing thiazole moieties were achieved in satisfactory overall
yields, although none of the synthetic steps was optimized. Refer-
ence compound 11 was obtained via convergent synthesis
(Scheme 1). First, compound 8 was converted into methyl oxime 9
in analogy to the method described for the preparation of intermedi-
ate 13. Compound 10 was obtained according to the procedure pre-
viously described.20 2-(Fluoromethyl)-4-(ethynyl)thiazole in turn
was obtained from 10 in situ by addition of TBAF to the Sonogashira
reaction mixture. 2-(Fluoromethyl)-4-(ethynyl)thiazole was then
coupledto thetrans-isomerof 9toaffordendproduct11in11%yield.
In a similar manner, 2-bromothiazole (12) was coupled to starting
material 13,24 which was prepared according to the procedure re-
cently described,26 to afford 14 in moderate yield (Scheme 2). The
2.2. In vitro binding assays
Compounds 11, 14, 24, 25 (Table 1) were investigated for their
binding affinity towards mGluR5. The four compounds exhibited
binding affinities in the nanomolar range. The fluoromethyl-thia-
zole analogue, 11, gave a Ki value of 5.5 1.1 nM (Fig. 2), whereas
Scheme 1. Synthesis of FTECMO (11, (E)-3-((2-(fluoromethyl)thiazol-4-yl)ethynyl)cyclohex-2-enone O-methyl oxime). Reagents and conditions: (a) NH2OCH3ÁHCl, pyridine,
rt, 18 h, 63%; (b) CuI, Pd(PPh3)4, DMF, Et3N, TBAF, rt, 20 h, 11%.

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Table 1
Binding affinity of four thiazole derivatives 11, 14, 24 and 25 towards mGluR5
Ligand
Structure of the compounds
Ki (nM)
11
5.5 1.1
14
24
25
20.3 6.3
36.0 11.1
21.9 7.0
2.4. Determination of log DpH7.4
The lipophilicity of [18F]-FTECMO was determined by the shake
flask method at physiological pH in analogy to the method de-
scribed by Wilson et al.27 A log DpH7.4 of 1.6 0.2 was obtained
for [18F]-FTECMO and this value compares favorably to the calcu-
lated log P value of 1.9.
Scheme 3. Syntheses of two thiazole analogues (24 and 25) of the mGluR5
radioligand 7 (Fig. 1). Reagents and conditions: (a) DMF, Pd(PPh3)4, CuI, Et3N, rt,
24 h, 24–54%; (b) DMF, NaH, (2-(2-bromoethoxy)ethoxy)(tert-butyl)dimethylsilane,
rt, 2 h, 72–80%; (c) (1) THF, TBAF, rt, 1.5 h; (2) CH2Cl2, toluenesulfonylchloride, 0 °C,
rt, 12 h, 85–91%; (d) THF, TBAF, 60 °C, 5 h, 25–28%.
the comparable 2-yl-thiazole analogue 14, lacking a methyl group
on the thiazole ring, exhibited a slightly reduced affinity with a Ki
value of 20.3 6.3 nM. Compounds 24 and 25 with ethoxy side
chain substitutions at the oxime moiety exhibited lower binding
affinities towards mGluR5. The desmethyl analogue 24 gave a Ki
value of 36.0 11.1 nM, while compound 25 showed a slightly
higher affinity (Ki = 21.9 7.0 nM) for mGluR5. Since compound
11 displayed the highest binding affinity of all the four candidates
tested, it was selected for radiolabeling and further evaluation.
2.5. In vitro stability
Stability studies in rat and human plasma revealed no radioac-
tive degradation products of [18F]-FTECMO at 37 °C after an incu-
bation period to 120 min. In rat liver microsomes, 93% of the
parent compound was still intact after 60 min of incubation. One
minor radioactive degradation product, more polar than the parent
compound, was detected by HPLC.
2.3. Radiosynthesis of [18F]-FTECMO
2.6. In vitro autoradiography
Incubation of rat brain slices with [18F]-FTECMO using two dif-
ferent concentrations (0.5 nM or 5 nM) resulted in a heterogeneous
binding of the tracer with the highest accumulation in mGluR5-
rich brain regions such as the hippocampus, striatum, and cortex
(Fig. 4). As expected, accumulation of radioactivity in the cerebel-
lum was negligible. Furthermore, blocking studies in which the
rat brain slices were incubated with [18F]-FTECMO together with
an excess of unlabeled ABP688 (500 nM) led to substantially re-
duced and homogeneous accumulation of activity in the rat brain
slices.
A one-step radiolabeling procedure was successfully applied for
the labeling of [18F]-FTECMO with fluorine-18 (Scheme 5). The
reaction proceeded well in MeCN at 90 °C in a reaction time of
10 min and the final product was obtained in a radiochemical yield
of up to 45% (decay corrected). The total synthesis time (from end
of bombardment) including the formulation of [18F]-FTECMO was
60 min. Radiochemical purity was greater than 99% (Fig. 3) and
the specific radioactivity ranged from 50 to 120 GBq/lmol
(n = 6). The identity of the radioligand was confirmed by co-injec-
tion with reference compound 11.
Scheme 4. Synthesis of the labeling precursor (E)-3-((2-(bromomethyl)thiazol-4-yl)ethynyl)cyclohex-2-enone O-methyl oxime (29). Reagents and conditions: (a) DMF, Et3N,
Pd(PPh3)4, CuI, rt, 24 h, ꢀ91% (crude); (b) THF, TBAF in THF (1 M), rt, 50 min, 47%; (c) THF, CBr4, P(Ph)3, rt, 2 h, 28%.

C. A. Baumann et al. / Bioorg. Med. Chem. 18 (2010) 6044–6054
6047
Figure 2. Displacement binding curve of FTECMO (11).
2.7. PET study
PET studies in male Wistar rats (n = 3) were undertaken to ana-
lyze whether mGluR5-expressing regions in the rat brain could be
visualized by [18F]-FTECMO. Summed images for the total acquisi-
tion time of 60 min did not show any substantial brain uptake in
the three rats tested. However, high skeletal accumulation of
radioactivity was evident (Fig. 5). Region of interest (ROI) analysis
Scheme 5. Radiosynthesis of [18F]-FTECMO. Reagents and conditions: (a) K[18F]F-
K2.2.2, ACN, 90 °C, 10 min.
Figure 3. (A) UV HPLC profile of formulated [18F]-FTECMO containing ascorbic acid (retention time = 2.17 min) (B) RadioHPLC profile of formulated [18F]-FTECMO (retention
time = 6.48 min).

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C. A. Baumann et al. / Bioorg. Med. Chem. 18 (2010) 6044–6054
Figure 4. In vitro autoradiography of horizontal sections of rat brains incubated under baseline (A1 and B1) and blocking conditions (A2 and B2).
of dynamic PET data of two rats confirmed the high radioactivity
uptake in the skeleton, which strongly increased during the scan-
ning period (Figs. 5 and 6). Radioactivity uptake in the brain was
low during the whole scan period. The dynamic data also showed
that radioactivity accumulation was higher in the hindbrain than
in the forebrain regions (cortex, striatum, hippocampus).
to [18F]-FTECMO. It accounted for 60% (in the sample withdrawn
at 5 min pi) and 31% (30 min pi) of the total radioactivity.
3. Discussion
MGluR5 has been shown to be involved in numerous central
nervous system disorders. Non-invasive in vivo imaging of the
receptor using PET can give further insight into the underlying
pathophysiological processes and help understand mGluR5 phar-
macology, which in turn will speed up drug development.28,29 Ef-
forts have been made in recent years to develop suitable mGluR5
PET tracers. However, to date only two mGluR5 PET ligands,
2.8. In vivo metabolism
Plasma samples were collected from the tail vein of a Wistar rat
at 5 and 30 min after [18F]-FTECMO iv injection. For both samples,
two radioactivity peaks were identified by HPLC and UPLC. The first
peak, which was absent before injection, appeared in the void vol-
ume, suggesting the in vivo formation of [18F]-fluoride or another
very hydrophilic radiometabolite. The second peak corresponded
[
11C]-ABP688 and [18F]SP203, have been fully characterized in hu-
mans. Although [11C]-ABP688 displays favorable in vivo imaging
parameters, the labeling with the short-lived nuclide carbon-11
Figure 5. MIP (maximum intensity projection) PET images of a rat brain obtained with [18F]-FTECMO.

C. A. Baumann et al. / Bioorg. Med. Chem. 18 (2010) 6044–6054
6049
mGluR5-rich brain regions. In contrast, a strong accumulation of
activity was observed in the bones, including the skull. ROI analysis
of the dynamic PET data confirmed uptake of radioactivity into the
bones and demonstrated that activity in the forebrain (cortex, stri-
atum, hippocampus) and the hindbrain (including cerebellum)
peaked at a low level immediately after radiotracer injection.
Based on the distribution pattern of mGluR5 in the mammalian
brain, radioactivity accumulation would be expected to be higher
in the forebrain than in the hindbrain. However, in all the PET
scans the uptake of radioactivity was higher in the hindbrain than
in the forebrain regions.
Since metabolism studies revealed only one radioactive metab-
olite, which is presumably [18F]-fluoride, it is unlikely that the
higher uptake resulted from a metabolite. It is also unlikely that
the higher accumulation in the hindbrain is derived from specific
binding to a target other than mGluR5 since specific binding usu-
ally results in much higher uptake values and differently shaped
time-activity curves. Moreover, the in vitro autoradiographic stud-
ies clearly indicated the specificity of [18F]-FTECMO for mGluR5-
rich brain regions when ABP688, a known ligand for mGluR5,
was used as a blocking agent. Finally, the discrepancy may result
from perfusion phenomena that cannot be properly addressed in
a brain PET scan, that is, in addition flawed by accumulation of
radioactivity in the skull. This high uptake of radioactivity in bones,
including the skull and in particular joints is a strong evidence for
in vivo radiodefluorination.32 This is in agreement with our find-
ings from the in vivo metabolism studies.
Figure 6. Time activity curves of [18F]-FTECMO uptake. ROI analysis of dynamic PET
scans from 0 to 60 min after i.v. injection of 30 MBq [18F]-FTECMO into male Wistar
rats. ROIs were defined for forebrain (cortex, striatum,hippocampus), hindbrain
(including cerebellum) and bone (joint). Mean dose-normalized values of two
independent experiments with error bars indicating the single values.
(t1/2 = 20 min) limits its widespread use. [18F]SP203 showed spe-
cies dependent in vivo defluorination in monkeys20 and in rats,30
that is, undesirable for in vivo imaging. However, enzymatic deflu-
orination was negligible in human studies.23,31
With the hope of obtaining a fluorine-18 labeled PET tracer with
structural elements of both [18F]SP203 and [11C]-ABP688, four novel
thiazole containing ABP688 derivatives (11, 14, 24, and 25) were
synthesized and screened for their binding affinity towards mGluR5.
Compound 14 does not contain a fluorine atom and served only as a
model compound. Compound 11, named FTECMO, exhibited a Ki va-
lue of 5.5 1.4 nM which is comparable with the Ki value of 4.4 1.0
nM for ABP688 obtained in a parallel displacement experiment. The
replacement of the methyl-pyridine moiety in ABP688 with a fluo-
romethyl-thiazole moiety was obviously well tolerated by mGluR5.
However, this does not hold true for compounds 24 and 25 (Table 1),
analogues of compound 7.26 Compound 7 was previously shown to
be a high affinity mGluR5 ligand. For these compounds, the binding
affinities were reduced by 7- and 10-fold, respectively, when the
pyridine ring was replaced with either a thiazole or a methylthiazole
moiety. The more promising candidate FTECMO, with the highest
binding affinity determined in this study, was selected for further
evaluation as a potential PET tracer. A convenient one-step radiola-
beling was established for [18F]-FTECMO. Reacting the bromo-
precursor 29 with K[18F]F-K222 in acetonitrile at 90 °C afforded
[
18F]-FPECMO, a fluoropyridine analogue of ABP688, similarly
showed no clear-cut visualization of mGluR5-rich brain regions
in rats. [18F]-FPECMO was also rapidly defluorinated in vivo and re-
sulted in radioactivity accumulation in bones and the skull.24 The
radioactivity uptake into rat brain after [18F]-FPECMO injection
peaked early after tracer injection but was eliminated from the
fore- and hindbrain regions rapidly. In the present study [18F]-
FTECMO showed substantially a lower radioactivity peak. The
visualization of mGluR5-rich brain regions with [18F]-FTECMO
in vivo was not only affected by radiodefluorination but it also
failed, because radioactive uptake in the brain was not sufficient
enough to yield a clear PET signal with an appropriate signal-to-
background ratio even at early time points (Fig. 5). The lipophilicity
of [18F]-FTECMO (log DpH7.4 = 1.6 0.2), however, suggests ideal
physicochemical properties for diffusion through the blood–brain
barrier.33 The low brain uptake cannot be assigned exclusively to
the rapid radiodefluorination of [18F]-FTECMO, since the amount
of remaining intact tracer—31% at 30 min pi—would be high en-
ough for the visualization of mGluR5-rich brain regions. The
in vivo metabolism of [11C]-ABP688, for example, follows a similar
time course with only 29% of parent tracer available at 30 min pi
but still enables good PET imaging.34 Presumably, [18F]-FTECMO
is hindered from passing the blood–brain barrier. The low penetra-
tion of [18F]-FTECMO may be due to low passive diffusion across
the cell membranes, although sufficient passive diffusion would
be expected based on its log D at pH 7.4. Alternatively, blood–brain
barrier penetration may be low because of efflux transport by P-
glycoprotein35 or another ABC transporter or a solute carrier.
Structurally, [18F]-FTECMO is similar to [18F]SP203. The enzy-
matic degradation of [18F]SP203 was reported to occur in the rat
brain and in the periphery, whereby the mechanism of radiodeflu-
orination was shown to be mediated by glutathionylation through
the glutathione-S-transferase system.30 Glutathione-S-transferases
are known to dehalogenate alkyl chlorides, bromides, and io-
dides.36 Due to the structural similarity of the [18F]-fluorothiazole
moiety in both tracers and the absence of substantial defluorina-
tion in microsomes (no glutathione was added), we assume that
the radiodefluorination in [18F]-FTECMO is most likely derived
from similar metabolic interactions.
[
18F]-FTECMO in good radiochemical yields (up to 45%, decay
corrected). Semi-preparative HPLC purification resulted in a highly
pure product with a radiochemical purity greater than 97% (Fig. 3).
Furthermore, high specific radioactivities ranging from 80 to
120 GBq/lmol were reproducibly obtained. The tracer was identi-
fied by co-injection with unlabeled FTECMO.
In vitro autoradiographical studies using rat brain slices re-
vealed a heterogeneous and displaceable binding of [18F]-FTECMO
in brain regions such as the hippocampus, striatum, and cortex, re-
gions known to contain high densities of mGluR5. In addition,
[
18F]-FTECMO exhibited high stability in human and in rat plasma
over 120 min. In rat liver microsomes, only minimal degradation
was observed when UPLC analysis was performed. The short reten-
tion time in the UPLC system points to a very hydrophilic degrada-
tion product, which is presumably [18F]-fluoride. The promising
results of the stability studies and the positive in vitro results such
as high binding affinity and displaceable accumulation of the tracer
in rat brain slices prompted us to further evaluate [18F]-FTECMO.
PET experiments were carried out in Wistar rats in order to
evaluate the suitability of [18F]-FTECMO in vivo. Despite an optimal
log D of 1.6, low accumulation of [18F]-FTECMO was observed in

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C. A. Baumann et al. / Bioorg. Med. Chem. 18 (2010) 6044–6054
It is noteworthy that in 2008, Brown et al.23 described the suc-
After stirring at rt for 18 h, 30 mL of H2O were added and the reac-
tion mixture was extracted with ether. The combined organic lay-
ers were washed with saturated CuSO4 solution (3 Â 30 mL) and
brine (20 mL) and dried over Na2SO4. Evaporation led to 1.6 g of
brown oil. Purification by flash column chromatography pentane/
EtOAc (95:5) gave the trans-isomer as yellowish crystals (802 mg,
63%). trans-isomer: 1H NMR (400 MHz, CDCl3): d 6.50 (s, 1H),
3.87 (s, 3H), 2.60 (t, J = 6.6 Hz, 2H), 2.52 (t, J = 6.3 Hz, 2H), 1.84
(quint, J = 4.1 Hz, 2H). MS m/z 203.85 (M+H)+. cis-isomer: 1H
NMR (400 MHz, CDCl3): d 7.12 (s, 1H), 3.84 (s, 3H), 2.65 (t,
J = 6.0 Hz, 2H), 2.36 (t, J = 6.0 Hz, 2H), 1.91 (quint, J = 6.6 Hz, 2H).
cessful in vivo imaging of [18F]SP203 in humans, which was not hin-
dered by in vivo defluorination. The striking species differences in
defluorination were presumably the result of species differences
in the expression, activity, and substrate selectivity of the mamma-
lian glutathione-S-transferases.37 In analogy to the metabolic profile
of [18F]SP203, the radiodefluorination of the [18F]-fluoromethy-
thiazole moiety in [18F]-FTECMO might be dramatically reduced in
primates compared to rats and thus the evaluation of [18F]-FTECMO
in higher species such as monkeys and humans may be warranted
once efflux transport at the blood–brain barrier is excluded.
5.2.2. (E)-3-((2-(Fluoromethyl)thiazol-4-yl)ethynyl)cyclohex-2-
enone O-methyl oxime (11)
4. Conclusion
Et3N (0.5 mL) and CuI (7.7 mg, 0.04 mmol) were added under
argon to a degassed solution of Pd(PPh3)4 (20.4 mg, 0.018 mmol)
and 10 (110 mg, 0.54 mmol) in DMF (5 mL). TBAF (1.2 mmol;
1.0 M solution in THF, 1.2 mL) was added to a solution of 2-(fluo-
romethyl)-4-((trimethylsilyl)ethynyl)thiazole (85 mg, 0.6 mmol)
in DMF (4 mL). After stirring for 30 min, the mixtures were com-
bined and stirred for 20 h at rt. Sat. NH4Cl solution (20 mL) was
added and the resulting mixture was extracted with EtOAc
(3 Â 20 mL). The combined organic layers were washed with water
(2 Â 20 mL) and brine (10 mL) and dried over Na2SO4. Evaporation
of the solvents under reduced pressure gave 370 mg of brown
crude material. The crude product was purified twice by column
chromatography with pentane/EtOAc (8:2) to afford compound
11 as brown crystals (17 mg, 11%). Mp: 58.5 °C. 1H NMR
(400 MHz, CDCl3): d 7.53 (s, 1H), 6.54 (s, 1H), 5.62 (d, J = 47.1 Hz,
2H), 3.92 (s, 3H), 2.54 (t, J = 6.3 Hz, 2H), 2.37 (t, J = 5.8 Hz, 2H),
1.79 (quint, J = 6.3 Hz, 2H). 13C NMR (100 MHz, CDCl3): d 20.8,
22.2, 29.4, 62.0, 80.6 (d, J = 170.6 Hz), 86.0, 90.3, 123.9 (d,
J = 2.7 Hz), 127.0, 130.6, 137.6, 155.3, 164.8 (d, J = 24.3 Hz). 19F
NMR (375 MHz, CDCl3): d À211.52. MS m/z 264.94 (M+H)+. HRMS
calcd for C13H13FN2OS, 264.0728; found, 264.0728.
In summary, the syntheses and investigation of four novel thia-
zole containing ABP688 analogues led to the identification of a
high affinity ligand for mGluR5, FTECMO. Its radiolabeled analogue,
[
18F]-FTECMO, represents a combination of structural elements
from
[ [
18F]SP203 and 11C]-ABP688, that displayed favorable
in vitro characteristics. [18F]-FTECMO is however not suitable for
mGluR5 imaging in vivo in rats. The further evaluation of [18F]-
FTECMO in higher species such as monkeys and humans may shed
more light on the in vivo utility of this new PET ligand.
5. Experimental
5.1. General
Reagents and solvents utilized for experiments were obtained
from commercial suppliers (Sigma–Aldrich, Alfa Aesar, Merck and
Flucka) and were used without further purification unless stated
otherwise. [3H]-M-MPEP was provided by Novartis. Thin layer
chromatography performed on pre-coated Silica Gel 60 F245 alu-
minum sheets suitable for UV absorption detection of compounds
was used for monitoring reactions. Nuclear magnetic resonance
spectra were recorded with a Bruker 400 MHz spectrometer with
an internal standard from solvent signals. Chemical shifts are given
in parts per million (ppm) relative to tetramethylsilane (0.00 ppm).
Values of the coupling constant, J, are given in hertz (Hz). The fol-
lowing abbreviations are used for the description of 1H NMR spec-
tra: singlet (s), doublets (d), triplet (t), quartet (q), quintet (quint),
doublet of doublets (dd), multiplet (m). The chemical shifts of com-
plex multiplets are given as the range of their occurence. Low-res-
olution mass spectra (LR-MS) were recorded with a Micromass
Quattro micro API LC-ESI. High-resolution mass spectra (HR-MS)
were recorded with a Bruker FTMS 4.7T BioAPEXII (ESI). Quality
control of the final products was performed on an Agilent 1100
system equipped with a radiodetector from Raytest using a re-
5.2.3. (E)-3-(Thiazol-2-ylethynyl)cyclohex-2-enone O-methyl
oxime (14)
Pd(PPh3)4 (15 mg, 0.013 mmol) was added under argon to a de-
gassed solution of 2-bromothiazole 12 (200 mg, 1.22 mmol) in
DMF (2.5 mL). The mixture was stirred at rt for 5 min before Et3N
(1 mL) was added. The mixture was stirred for further 5 min before
addition of CuI (6 mg, 0.03 mmol) and 13 (180 mg, 1.22 mmol),
that was obtained as previously described.26 The mixture was stir-
red at room temperature for 48 h, quenched with satd NH4Cl
(20 mL) and extracted with ether (3 Â 20 mL). The combined or-
ganic layers were washed with water (2 Â 20 mL) and brine
(20 mL). The solvents were evaporated under reduced pressure
and the residue was purified by column chromatography with pen-
tane/EtOAc (9:1) to afford compound 14 as light yellow crystals
(74 mg, 26%). Mp: 55.6 °C. 1H NMR (400 MHz, CDCl3): d 7.61 (d,
J = 3.2 Hz, 1H), 7.37 (d, J = 3.5 Hz, 1H), 6.60 (s, 1H), 3.93 (s, 3H),
2.55 (t, J = 6.0 Hz, 2H), 2.39 (t, J = 5.8 Hz, 2H), 1.81 (quint,
J = 6.7 Hz, 2H). 13C NMR (100 MHz, CDCl3): d 21.1, 22.4, 29.4,
62.5, 85.6, 94.9, 121.3, 126.6, 132.2, 144.1, 148.9, 155.4. MS m/z
232.94 [M+, 100]. MS m/z 232.94 (M+H)+. HRMS calcd for
versed phase column (Gemini 10
l
m C18, 300 Â 3.9 mm, Phenom-
enex) by applying an isocratic solvent system with 70% MeCN in
water and a flow of 1 mL/min. In vitro stability tests were per-
formed on a Waters Acquitiy UPLC system (Berthold radiodetector)
occupying an Acquitiy UPLC BEH C18 1.7
l
m (2.1 Â 50 mm) col-
umn. A binary solvent system with acetonitrile (solvent A) and
water/acetonitrile (9:1) (solvent B) at a flow rate of 0.7 mL/min
was used. During the first three minutes a gradient (100% B to
100% A) was applied followed by 0.5 min under isocratic condi-
tions (100% A). The Swiss Federal Veterinary Office approved ani-
mal care and all experimental procedures.
C12H13N2OS, 233.0743; found, 233.0744.
5.2.4. (E)-3-(Thiazol-4-ylethynyl)cyclohex-2-enone oxime (18)
Compound 18 was prepared in an analogous way to compound
14. Starting from 15 (1000 mg, 6.1 mmol) and 17 (840 mg,
6.2 mmol) compound 18 was obtained as yellow crystals
(720 mg, 54%). The product was purified by column chromatogra-
phy using pentane/EtOAc (1:1) as the mobile phase. 1H NMR
(400 MHz, CDCl3): d 8.80 (s, 1H), 7.53 (s, 1H), 6.61 (s, 1H), 2.63 (t,
J = 6.8 Hz, 2H), 2.40 (t, J = 5.7 Hz, 2H), 1.83 (quint, J = 6.2 Hz, 2H).
5.2. Chemistry
5.2.1. 3-Bromocyclohex-2-enone O-methyl oxime (9)
O-Methylhydroxylamine hydrochloride (0.8 g, 9.4 mmol) was
added to a solution of 8 (1.09 g, 6.27 mmol) in pyridine (20 mL).

C. A. Baumann et al. / Bioorg. Med. Chem. 18 (2010) 6044–6054
6051
13C NMR (100 MHz, CDCl3): d 20.7, 21.6, 29.3, 86.4, 90.3, 122.4,
127.6, 130.4, 138.3, 152.8, 156.3. MS m/z 218.82 (M+H)+.
tracted with ether (3 Â 20 mL). The combined organic layer was
washed with water (2 Â 20 mL) and brine (20 mL), dried over
Na2SO4 and the solvents were evaporated under reduced pressure.
The residue was purified by column chromatography with pen-
tane/Et2O (1:4) to give pure 22 as a yellow oil (360 mg, 91%). 1H
NMR (400 MHz, CDCl3): d 8.78 (s, 1H), 7.80 (d, J = 8.3 Hz, 2H),
7.52 (s, 1H), 7.34 (d, J = 10.2 Hz, 2H), 6.52 (s, 1H), 4.20–4.14 (m,
4H), 3.72–3.65 (m, 4H), 2.55 (t, J = 6.8 Hz, 2H), 2.44 (s, 3H), 2.38
(t, J = 5.2 Hz, 2H), 1.79 (quint, J = 6.6 Hz, 2H). 13C NMR (100 MHz,
CDCl3): d 20.8, 21.6, 22.3, 29.3, 68.7, 69.2, 69.8, 73.4, 86.4, 90.3,
122.4, 127.5, 128.0, 129.8, 130.3, 133.1, 138,4, 144.8, 152.6,
155.8. MS m/z 482.71 (M+Na)+. HRMS calcld for C22H24N2NaO5S2,
483.1019; found, 483.1012.
5.2.5. (E)-3-((2-Methylthiazol-4-yl)ethynyl)cyclohex-2-enone
oxime (19)
This compound was prepared analogously to compound 18.
Starting from 16 (790 mg, 4.44 mmol) and 17 (600 mg, 4.44 mmol),
compound 19 was obtained as yellow crystals (248 mg, 24%). Mp:
147.8–148.4 °C. 1H NMR (400 MHz, CDCl3): d 7.21 (s, 1H), 6.59 (s,
1H), 2.73 (s, 3H), 2.64 (t, J = 6.1 Hz, 2H), 2.41 (t, J = 6.2 Hz, 2H),
1.83 (quint, J = 6.3 Hz, 2H). 13C NMR (100 MHz, CDCl3): d 19.2,
20.6, 21.6, 29.4, 86.6, 89.4, 122.9, 125.4, 129.3, 136.6, 156.6,
165.9. MS m/z 232.82 (M+H)+. HRMS calcld for C12H12N2OS,
232.0670; found, 232.0665.
5.2.9. (E)-2-(2-(3-((2-Methylthiazol-4-yl)ethynyl)cyclohex-2-
enylideneaminooxy)ethoxy)ethyl 4-methylbenzenesulfonate
(23)
5.2.6. (E)-3-(Thiazol-4-ylethynyl)cyclohex-2-enone O-2-(2-(tert-
butyldimethylsilyloxy)ethoxy)ethyl oxime (20)
To a solution of 18 (250 mg, 1.15 mmol) in DMF (21 mL) was
added 60% NaH (90 mg, 2.25 mmol). After stirring for 30 min at
rt, (2-(2-bromoethoxy)ethoxy)(tert-butyl)dimethylsilane (340 mg,
1.2 mmol) was added. The reaction mixture was stirred for 1.5 h
This compound was synthesized in analogy to compound 22.
Starting from 21 (290 mg, 0.66 mmol), compound 23 was obtained
as a yellow oil (270 mg, 85%). 1H NMR (400 MHz, CDCl3): d 7.80 (d,
J = 8.1 Hz, 2H), 7.33 (d, J = 8.1 Hz, 1H), 7.31 (s, 1H), 6.49 (s, 1H),
4.18–4.14 (m, 4H), 3.70–3.65 (m, 4H), 2.72 (s, 3H), 2.53 (t,
J = 6.5 Hz, 2H), 2.44 (s, 3H), 2.36 (t, J = 6.5 Hz, 2H), 1.78 (quint,
J = 6.5 Hz, 2H). 13C NMR (100 MHz, CDCl3): d 19.2, 20.8, 21.6, 22.3,
29.4, 68.7, 69.2, 69.8, 73.4, 86.8, 89.6, 122.6, 127.6, 128.0, 129.8,
130.1, 133.1, 136.7, 144.8, 155.9, 165.8. MS m/z 474.84 (M+H)+.
HRMS calcd for C23H26N2O5S2H+, 475.1356; found, 475.1356.
and then quenched by addition of
a 50% NaHCO3 solution
(15 mL). The mixture was extracted with ether (3 Â 20 mL). The
combined organic layers were washed with water (2 Â 20 mL)
and brine (20 mL) and dried over Na2SO4. The solvents were evap-
orated under reduced pressure and the obtained crude product
was purified by column chromatography with pentane/EtOAc
(6:4) to give compound 20 as a slightly yellow oil (390 mg, 80%).
1H NMR (400 MHz, CDCl3): d 8.78 (s, 1H), 7.51 (s, 1H), 6.53 (s,
1H), 4.25 (t, J = 5.4 Hz, 2H), 3.76 (q, J = 5.40 Hz, 4H), 3.56 (t,
J = 4.9 Hz, 2H), 2.57 (t, J = 7.0 Hz, 2H), 2.38 (t, J = 6.5 Hz, 2H), 1.79
(quint, J = 7.0 Hz, 2H), 0.89 (s, 9H), 0.06 (s, 6H). 13C NMR
(100 MHz, CDCl3): d À5.3, 18.4, 20.8, 22.3, 25.9, 29.4, 62.8, 69.7,
72.7, 73.7, 86.3, 90.3, 122.4, 127.2, 130.5, 138.4, 152.6, 155.6. MS
m/z 420.90 (M+H)+. HRMS calcld for C21H32N2NaO3SSi, 443.1795;
found, 443.1805.
5.2.10. (E)-3-(Thiazol-4-ylethynyl)cyclohex-2-enone O-2-(2-
fluoroethoxy)ethyl oxime (24)
Hydrated TBAF (280 mg, 0.89 mmol) was dried under high vac-
uum at 45 °C for 24 h and was then dissolved in dry THF (10 mL). A
solutionof 22(180 mg, 0.4 mmol) indry THF (5 mL)was added. After
stirring the mixture for 5 h at 60 °C, water (10 mL) was added. The
solution was extracted with ether (3 Â 20 mL). The organic layers
were washed with water (2 Â 20 mL) and brine (20 mL) and dried
over Na2SO4. After evaporation of the solvents, the brown crude
productwaspurified bycolumn chromatography withEt2O/pentane
(6:1) to give compound 24 as a clear oil (26 mg, 21%). 1H NMR
(400 MHz, CDCl3): d 8.78 (s, 1H), 7.52 (s, 1H), 6.53 (s, 1H), 4.62 (t,
J = 4.1 Hz, 1H), 4.50 (t, J = 4.2 Hz, 1H), 4.27 (t, J = 5.1 Hz, 2H), 3.81–
3.76 (m, 3H), 3.71 (t, J = 3.3 Hz, 1H), 2.57 (t, J = 6.8 Hz, 2H), 2.38 (t,
J = 6.3 Hz, 2H), 1.80 (quint, J = 6.6 Hz, 2H). 13C NMR (100 MHz,
CDCl3): d 20.8, 22.3, 29.4, 69.8, 70.3 (d, J = 19.4 Hz), 73.5, 83.1 (d,
J = 169.1 Hz), 86.3, 90.3, 122.4, 127.4, 130.4, 138.4, 152.6, 155.8.
19F NMR (376 MHz, CDCl3): d À223.09. MS m/z 308.80 (M+H)+.
5.2.7. (E)-3-((2-Methylthiazol-4-yl)ethynyl)cyclohex-2-enone O-
2-(2-(tert-butyldimethylsilyloxy)ethoxy)ethyl oxime (21)
This compound was prepared analogously to compound 20.
Starting from 19 (233 mg, 1.0 mmol) and 2-(2-bromoethoxy)(t-
butyl)dimethylsilane (348 mg, 1.2 mmol), compound 21 was ob-
tained as a clear oil (315 mg, 72%). 1H NMR (400 MHz, CDCl3): d
7.31 (s, 1H), 6.51 (s, 1H), 4.25 (t, J = 5.0 Hz, 2H), 3.78–3.73 (m,
4H), 3.56 (t, J = 5.1 Hz, 2H), 2.72 (s, 3H), 2.56 (t, J = 6.1 Hz, 2H),
2.37 (t, J = 5.7 Hz, 2H), 1.78 (quint, J = 6.8 Hz, 2H), 0.89 (s, 9H),
0.07 (s, 6H). 13C NMR (100 MHz, CDCl3): d À5.3, 18.4, 19.2, 20.8,
22.3, 25.9, 29.4, 62.8, 69.8, 72.7, 73.7, 86.6, 89.7, 122.5, 127.3,
130.3, 136.7, 155.7, 165.8. MS m/z 434.96 (M+H)+. HRMS calcd
for C22H35N2O3SSi+, 435.2132; found, 435.2139.
5.2.11. (E)-3-((2-Methylthiazol-4-yl)ethynyl)cyclohex-2-enone
O-2-(2-fluoroethoxy)ethyl oxime (25)
This compound was obtained in an analogous way to 24. Start-
ing material 23 (150 mg, 0.32 mmol) gave a slightly yellow oil
(39 mg, 40%). 1H NMR (400 MHz, CDCl3): d 7.31 (s, 1H), 6.50 (s,
1H), 4.62 (t, J = 4.4 Hz, 1H), 4.50 (t, J = 3.9 Hz, 1H), 4.26 (t,
J = 5.1 Hz, 2H), 3.8 (t, J = 4.1 Hz, 3H), 3.71 (t, J = 4.3 Hz, 1H), 2.71
(s, 3H), 2.56 (t, J = 6.36 Hz, 2H), 2.36 (t, J = 7.0 Hz, 2H), 1.78 (quint,
J = 6.2 Hz, 2H). 13C NMR (100 MHz, CDCl3): d 19.2, 20.8, 22.3, 29.4,
69.9, 70.3 (d, J = 20.5 Hz), 73.5, 83.0 (d, J = 169.1 Hz), 86.8, 89.6,
122.5, 127.5, 130.2, 136.7, 155.8, 165.8. 19F NMR (376 MHz, CDCl3):
d À223.11. MS m/z 322.77 (M+H)+. HRMS calcd for C16H19FN2-
NaO2S+, 345.1044; found, 345.1044.
5.2.8. (E)-2-(2-(3-(Thiazol-4-ylethynyl)cyclohex-2-
enylideneaminooxy)ethoxy)ethyl 4-methylbenzenesulfonate
(22)
To a solution of 20 (363 mg, 0.86 mmol) in THF (22 mL) was
added TBAF trihydrate (680 mg, 2.15 mmol). After stirring the
mixture for 1.5 h at rt, water (15 mL) was added. The solution
was extracted with EtOAc and the organic layers were washed with
water and brine and dried over Na2SO4. Evaporation of solvents un-
der reduced pressure led to an orange crude product which was
used directly without further purification. To the solution of (E)-
3-(thiazol-4-ylethynyl)cyclohex-2-enone O-2-(2-hydroxyethoxy)-
ethyl oxime (crude = 338 mg) in dry CH2Cl2 (6 mL) was added
5.2.12. (E)-3-((2-((tert-Butyldimethylsilyloxy)methyl)thiazol-4-
yl)ethynyl)cyclohex-2-enone O-methyl oxime (27)
Et3N (312
l
L). The mixture was cooled to 0 °C and then treated
Compound 27 was prepared in an analogous way to compound
14. Starting from 13 (430 mg, 2.88 mmol) and 26 (950 mg,
2.67 mmol) compound 27 was obtained (720 mg, 54%).
with toluenesulfonylchloride (401 mg, 1.3 mmol). After stirring at
rt for 8 h, the mixture was diluted with water (25 mL) and ex-

6052
C. A. Baumann et al. / Bioorg. Med. Chem. 18 (2010) 6044–6054
Purification by silica gel flash column chromatography with
pentane/Et2O (9:1) led to a mixture containing the desired product.
It was used without further purification.
nM) as a radioligand. The membranes (see above) were incubated
with increasing concentrations of the test compound (1 pM to
100 lM), each in triplicate, in incubation buffer II (30 mM NaHE-
PES, 110 mM NaCl, 5 mM KCl, 2.5 mM CaCl2 Â H20, 1.2 mM MgCl2,
5.2.13. (E)-3-((2-(Hydroxymethyl)thiazol-4-yl)ethynyl)cyclohex-
2-enone O-methyl oxime (28)
pH 8). Non-specific binding was determined in the presence of
ABP688 (100
lM). The test samples with a total volume of
To a solution of 27 containing impurities (920 mg, 2.443 mmol)
in THF (25.5 mL) was added a solution of TBAF (1 M) in THF
(2.95 mL, 2.923 mmol). After stirring the mixture for 1 h at rt the
reaction was quenched with water (20 mL). The mixture was ex-
tracted with EtOAc (3 Â 20 mL) and the combined organic layers
were washed with water (2 Â 20 mL) and brine (20 mL) and dried
with Na2SO4. The solvents were evaporated under reduced pres-
sure and the residue was purified by flash column chromatography
with pentane/EtOAc (6:4) to give the desired alcohol 28 (304 mg,
yield = 47%). 1H NMR (400 MHz, CDCl3): d 7.43 (s, 1H), 6.49 (s,
1H), 4.94 (s, 1H), 3.90 (s, 3H), 3.36 (s, 1H), 2.52 (t, J = 6.5 Hz, 2H),
2.35 (t, J = 6.0 Hz, 2H), 1.78 (quint, J = 6.5 Hz, 2H). 13C NMR
(100 MHz, CDCl3): d 20.8, 22.1, 29.4, 62.0, 62.1, 86.5, 90.0, 123.0,
127.2, 130.3, 137.1, 155.4171.5. MS m/z 263.02 (M+H)+. HRMS
calcd for [M+] C13H14FN2O2S+, 262.0771; found: 262.0768.
200 L were incubated for 45 min at rt. For the removal of free
l
radioligand, 4 mL of ice-cold incubation buffer II were added fol-
lowed by vacuum filtration over GF/C filters (Whatman) that were
previously impregnated with poly(ethyleneimine) 0.05% (Fluka
#03880, Switzerland). After rinsing the filters twice with 4 mL of
ice-cold buffer II, the filters were transferred to 4 mL scintilla-
tion-liquid (Ultima Gold, Perkin Elmer) in beta scintillation vials.
Radioactivity retained on the filters was measured by beta count-
ing (Liquid Scintillation Analyser 1900TR, Canberra Packard). Data
was evaluated with KELL Radlig Software (Biosoft). For calculation
of the Ki value, the Cheng–Prusoff equation was applied, with a KD
value of 2.0 nM for [3H]-M-MPEP.
5.4. Radiosynthesis of [18F]-FTECMO
The conversion of 29 into [18F]-FTECMO was achieved via nucle-
ophilic substitution with [18F]-fluoride. [18F]-fluoride was obtained
via the 18O(p,n)18F reaction using 98% enriched 18O-water. 18FÀ
was trapped from the aqueous solution on a light QMA cartridge
(Waters), which was previously preconditioned with 0.5 M K2CO3
(5 mL) and water (5 mL). A volume of 1 mL of Kryptofix K2.2.2. solu-
tion (Kryptofix K2.2.2.; 2.5 mg, K2CO3, 0.5 mg in MeCN/water (3:1))
was used for the elution of 18FÀ from the cartridge. The solvents
were evaporated at 110 °C under vacuum in the presence of slight
inflow of nitrogen gas. After addition of acetonitrile (1 mL), azeo-
tropic drying was carried out. This procedure was repeated twice
and gave rise to dry K2.2.2.–K[18F] F complex. Already 15 min after
5.2.14. (E)-3-((2-(Bromomethyl)thiazol-4-yl)ethynyl)cyclohex-
2-enone O-methyl oxime (29)
A solution of alcohol 28 (261 mg, 0.995 mmol) in benzene
(9.5 mL) was cooled to 0 °C and CBr4 (1.661 mg, 5.024 mmol) and
triphenylphosphine (1.312 mg, 5.0 mmol) were added. The mix-
ture was allowed to warm up to rt and stirred for 2 h. Then the
reaction mixture was filtered over Celite and the solvents were
evaporated under reduced pressure. Flash column chromatography
with hexane/EtOAc (5:1) as the mobile phase gave 29 as a beige so-
lid (89 mg, 30%). Mp: 78.9–79.2 °C. 1H NMR (400 MHz, CDCl3): d
7.49 (s, 1H), 6.52 (s, 1H), 4.70 (s, 2H), 3.91 (s, 3H), 2.53 (t,
J = 6.4 Hz, 2H), 2.37 (t, J = 6.2 Hz, 2H), 1.79 (quint, J = 6.5 Hz, 2H).
13C NMR (100 MHz, CDCl3): d 20.8, 22.1, 26.2, 29.3, 62.0, 86.1,
90.1, 124.8, 127.0, 130.6, 137.5, 155.3, 165.7. MS m/z 326.92
(M+H)+. HRMS calcd for [M+] C13H13BrN2OS+, 323.9927; found:
323.9930.
end of bombardment a solution of precursor 30 (2 mg in 300 lL
of dry acetonitrile) was added to the dried K2.2.2.–K[18F] F complex.
The reaction mixture was heated at 90 °C for 10 min, followed by
the addition of 50% MeCN in water (2 mL). Purification by semipre-
parative HPLC was carried out on a HPLC system equipped with a
Merck-Hitachi L-6200A intelligent pump, a Knauer Variable Wave-
length Monitor UV detector and a Geiger Müller LND 714 counter
with Eberlein RM-14 instrument using a reversed phase column
5.3. In vitro binding assays
5.3.1. Preparation of membranes
(Gemini 5
l
C18, 250 Â 10 mm, Phenomenex) with a solvent sys-
Sprague Dawley rats were sacrificed by decapitation and the
brains were harvested immediately. After removal of the cerebel-
lum the brain material was homogenized in 10 volumes of ice-cold
sucrose buffer (0.32 M sucrose; 10 mM Tris/acetate buffer pH 7.4)
with a polytron (PT-1200 C, Kinematica AG) for 1 min at setting 4.
The obtained homogenate was centrifuged (1000g, 15 min, 4 °C)
and the supernatant containing the membranes was removed
and kept while the pellet was resuspended in five volumes of su-
crose buffer. After a second round of homogenization and centrifu-
gation, the two supernatants were combined and centrifuged at
17,000g for 20 min at 4 °C to pellet the membranes. The pellet
was resuspended with incubation buffer (5 mM Tris/acetate buffer,
pH 7.4) and centrifuged again at 17,000g, (20 min, 4 °C) followed
by resuspension of the sedimented membranes with incubation
buffer. The final membrane suspension was stored in aliquots at
À70 °C. For each assay the required amount of membrane prepara-
tion was thawed and kept on ice during all preparation processes.
The protein concentration was determined with a Bio-Rad Micro-
assay with bovine serum albumin as a standard protein (Bradford,
1976).38
tem and gradient as follows: H2O (solvent A), acetonitrile (solvent
B); flow 5 mL/min; 0–10 min: 10% B, 10–20 min: 10–50% B, 20–
30 min: 50% B. [18F]-FTECMO was eluted approximately 50 min
after end of bombardment and ascorbic acid (16 mg) was added
to the fraction containing [18F]-FTECMO before the mixture was di-
luted with water (30 mL). The product was trapped on a C18 light
SepPak cartridge (Waters), which was preconditioned with ethanol
(5 mL) and water (5 mL). After washing the cartridge with 10 mL of
water for removal of traces of acetonitrile, the product was eluted
with 0.5 mL of ethanol through a sterile filter (0.2 lm). In order to
obtain an injectable solution, the fraction was diluted with sterile
saline water (9.5 mL) containing ascorbic acid (1 mg/mL). Determi-
nation of relative lipophilicity, radiochemical purity and specific
activity was carried out during the quality control step. Identifica-
tion of [18F]-FTECMO was achieved by co-injection of reference 11.
5.5. Determination of log DpH7.4
The shake flask method27 was performed (n = 5) with 0.5 mL of
1-octanol saturated with phosphate buffer (pH 7.4) and 0.5 mL of
phosphate buffer (pH 7.4) saturated with 1-octanol. After addition
5.3.2. Displacement assays
of 20
mixed for 15 min and then centrifuged at 5000g for 5 min before
l
L of [18F]-FTECMO solution (80 kBq) the samples were
For all four novel ligands (11, 14, 24 and 25) the binding affinity
was determined by displacement assays using [3H]-M-MPEP (2

C. A. Baumann et al. / Bioorg. Med. Chem. 18 (2010) 6044–6054
6053
the radioactivity in each phase was determined using a gamma
counter (Wizard, Perkin Elmer).
quired in list-mode and reconstructed in user-defined time frames
(dynamic: 5 Â 2, 4 Â 5, 3 Â 10 min, two animals; static: 1 Â 60
min, three animals) with a voxel size of 0.3775 Â 0.3775 Â 0.775
mm and a matrix size of 175 Â 175 Â 61. Image files were evalu-
ated by region-of-interest (ROI) analysis using the dedicated soft-
ware PMOD.41
5.6. In vitro stability tests
For the determination of the in vitro plasma stability of the radi-
oligand, 13.5
and rodent plasma (386.5
120 min at 37 °C. At five different time points (0, 30, 60, 90, and
120 min) aliquots (70 L) were taken and transferred into ice-cold
acetonitrile (140 L). After 10 min of centrifugation (5000g, 4 °C),
l
L of [18F]-FTECMO in ethanol were added to human
l
L). The solutions were incubated for
5.9. In vivo metabolism
l
For the determination of in vivo radiometabolites a male Wistar
l
rat was injected with [18F]-FTECMO via a lateral tail vein (160
lL,
the supernatant was analyzed by UPLC applying the conditions de-
200 MBq). Blood samples were withdrawn from the opposite vein
at 5 and 30 min post-injection. Whole blood was collected in hep-
arin-coated tubes (BD Vacutainers) and centrifuged at 5000g for
5 min. The proteins of the supernatant plasma were precipitated
by addition of an equal volume of acetonitrile and centrifuged at
5000g for 5 min. The supernatant was analyzed by analytical HPLC
and UPLC.
scribed above.
[
18F]-FTECMO microsomal stability was investigated employing
pooled rat liver microsomes (BD Bioscience). The test compound
(20–50 nM) or 15 M testosterone (positive control), was preincu-
l
bated for 5 min with 10 mM NADPH in 0.1 M phosphate buffer (pH
7.4) at 37 °C before addition of the microsome suspension
(0.52 mg/mL protein) or a suspension of boiled microsomes as a
negative control. At four different time points (0, 15, 30, and
Acknowledgments
60 min) 150 lL of ice-cold acetonitrile were added in order to pre-
cipitate all active enzymes. The samples were centrifuged at 5000g
for 5 min to obtain the supernatant to be analyzed by UPLC as
described.
We acknowledge the technical support of Claudia Keller, Petra
Wirth and Mathias Nobst. We thank Christophe Lucatelli and Har-
riet Struthers for fruitful discussion.
5.7. In vitro autoradiography
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