Structure-activity relationships of fluorinated (E)-3-((6-Methylpyridin-2- yl)ethynyl)cyclohex-2-enone- O -methyloxime (ABP688) derivatives and the discovery of a high affinity analogue as a potential candidate for imaging metabotropic glutamate recepors subtype 5 (mGluR5) with Positron Emission Tomography (PET)

Article abstract of DOI:10.1021/jm901850k

The metabotropic glutamate receptor subtype 5 (mGluR5) is recognized to be involved in numerous brain disorders. In an effort to obtain a fluorine-18 labeled analogue of the mGluR5 PET tracer [¹¹C]ABP688, 13 novel ligands based on the core structure of ABP688 were synthesized. Molecules in which the methyl group at the oxime functionality of ABP688 was replaced by fluorobenzonitriles, fluoropyridines, and fluorinated oxygen containing alkyl side chains were investigated. Substituents at the oxime functionality are well tolerated and resulted in five candidates with K_i values below 10 nM. The most promising candidate, (E)-3-(pyridin-2-ylethynyl)cyclohex-2-enone-O-2- (2-fluoroethoxy)ethyloxime (38, K_i = 3.8 nM), was radiolabeled with fluorine-18. Scatchard analysis of [¹⁸F]38 which modeled best for two sites pointed to high binding affinity (K_D1 = 0.61 ± 0.19 nM and K_D2 = 13.73 ± 4.69 nM) too. These data strongly suggest the further evaluation of [¹⁸F]38 as a candidate for imaging the mGluR5.

Full text of DOI:10.1021/jm901850k

J. Med. Chem. 2010, 53, 4009–4017 4009
DOI: 10.1021/jm901850k
Structure-Activity Relationships of Fluorinated (E)-3-((6-Methylpyridin-2-yl)ethynyl)cyclohex-
2-enone-O-methyloxime (ABP688) Derivatives and the Discovery of a High Affinity Analogue as a
Potential Candidate for Imaging Metabotropic Glutamate Recepors Subtype 5 (mGluR5) with
Positron Emission Tomography (PET)
Cindy A. Baumann,† Linjing Mu,† Sinja Johannsen, Michael Honer, Pius A. Schubiger, and Simon M. Ametamey*
Center for Radiopharmaceutical Sciences of ETH, PSI and USZ, Department of Chemistry and Applied Biosciences of ETH Zurich,
Switzerland, Wolfgang-Pauli Strasse 10, 8093 Zurich, Switzerland. †Both authors contributed equally.
Received December 15, 2009
The metabotropic glutamate receptor subtype 5 (mGluR5) is recognized to be involved in numerous
brain disorders. In an effort to obtain a fluorine-18 labeled analogue of the mGluR5 PET tracer
[11C]ABP688, 13 novel ligands based on the core structure of ABP688 were synthesized. Molecules in
which the methyl group at the oxime functionality of ABP688 was replaced by fluorobenzonitriles,
fluoropyridines, and fluorinated oxygen containing alkyl side chains were investigated. Substituents at
the oxime functionality are well tolerated and resulted in five candidates with Ki values below 10 nM.
The most promising candidate, (E)-3-(pyridin-2-ylethynyl)cyclohex-2-enone-O-2-(2-fluoroethoxy)-
ethyloxime (38, Ki = 3.8 nM), was radiolabeled with fluorine-18. Scatchard analysis of [18F]38 which
modeled best for two sites pointed to high binding affinity (KD1 = 0.61 ( 0.19 nM and KD2 = 13.73 (
4.69 nM) too. These data strongly suggest the further evaluation of [18F]38 as a candidate for imaging
the mGluR5.
Introduction
subdivided into three groups according to their sequence
homology, receptor pharmacology, and signal transduction
pathways. Group I consists of mGluR1 and mGluR5 that are
mainly located postsynaptically and activate phospholipase C
via a Gq protein, while group II mGluRs (mGluR2 and
mGluR3) and group III mGluRs (mGluR4, -6, -7, and -8)
are located presynaptically and are coupled to a Gi protein.1-3
The metabotropic glutamatereceptorshave beenimplicated
in numerous central nervous system (CNS) disorders. Espe-
cially, mGluR5 was shown in cell culture and in several animal
models to play a certain role for the development of neurode-
generative diseases like Alzheimer’s disease4,5 and Parkinson’s
disease6,7 or CNS disorders such as schizophrenia,8,9 depres-
sion, anxiety,10 neuropathic pain,11,12 drug addiction,13 and
fragile X syndrome.14 Although the underlying pathophysio-
logical processes are not yet well-understood, it is generally
agreed that mGluR5 is an important future drug target and
could provide a diagnostic target as well.15
The mGluR5 consists of a large extracellular N-terminus
sheltering the orthosteric glutamate binding site,16 the seven
transmembrane domains (TMs) typical for GPCRs and an
intracellular C-terminusthat interactswiththe G-protein. The
orthosteric binding site is highly conserved among glutamate
receptors, hampering the development of subtype-specific
ligands. Most of the effort is therefore spent on the development
of allosteric modulators with their binding site located within
the seven TMs of the receptor protein.17 The allosteric antago-
nist binding site of mGluR518 was investigated intensively with
2-methyl-6-(phenylethynyl)pyridine (MPEP, Figure 1, 1)12,18
and 2-((3-methoxyphenyl)ethynyl)-6-methylpyridine (M-MPEP,
Figure 1, 2),19,20 two prototype allosteric antagonists with high
The amino acid glutamate is the main excitatory neuro-
transmitter in the mammalian brain. Its transmission of
neuronal signals is mediated by a large family of different
glutamate receptors subdivided into two classes. On the one
hand ionotropic glutamate receptors (i.e., kainate, R-amino-
3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPAa),
and N-methyl-D-aspartate (NMDA) receptors) mediate in
general fast excitatory neurotransmission. On the other
hand, the eight metabotropic glutamate receptor subtypes
(mGluRs) discovered only from 1991 on modulate ionotropic
glutamate receptor activity and are known to conduct neuro-
transmission in a fine-tuned way. The mGluRs belong to the
family of G-protein-coupled receptors (GPCRs) and are
*To whom correspondence should be addressed. Phone: þ41 44 633
7463. Fax: þ41 44 633 1367. E-mail: simon.ametamey@pharma.ethz.ch.
a Abbreviations: AMPA, R-amino-3-hydroxy-5-methyl-4-isoxazole-
propionic acid; CDPPB, 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)-
benzamide; CNS, central nervous system; DMF, N,N-dimethyl-
formamide; ESI, electrospray ionization; Et2O, diethyl ether; Et3N,
triethylamine; EtOAc, ethyl acetate; F-MTEB, 3-fluoro-5-((2-methylthia-
zol-4-yl)ethynyl)benzonitrile;F-PEB, 3-fluoro-5-(pyridine-2-ylethynyl)ben-
zonitrile; GPCR, G-protein-coupled receptor; HEPES, 4-(2-hydroxyethyl)-
piperazine-1-ethanesulfonic acid sodium salt; HPLC, high performance
liquid chromatography; M-MPEP, 2-((3-methoxyphenyl)ethynyl)-
6-methylpyridine; HRMS, high resolution mass spectroscopy; LRMS,
low resolution mass spectroscopy; MeCN, acetonitrile; mGluR5,
metabotropic glutamate receptor subtype 5; MPEP, 2-methyl-6-(phenyl-
ethynyl)pyridine; NMDA, N-methyl-D-aspartate; PEG200, polyethylene
glycol 200; PET: positron emission tomography; rt, room temperature;
SAR, structure-activity relationship; TBAF, tetra-n-butylammonium
fluoride; TBS, tert-butyldimethylsilane; THF, tetrahydrofuran; TM,
transmembrane domain.
r
2010 American Chemical Society
Published on Web 04/22/2010
pubs.acs.org/jmc

4010 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Baumann et al.
Figure 1. Structures of mGluR5 antagonists and radioligands.
Scheme 1. Synthetic Pathway toward Compounds with Varying Positions of the Nitrogen Atom in the Pyridine Ring (Series 1
Compounds)a
a Reagents and conditions: (a) Pd(PPh3)4, CuI, Et3N, DMF, rt, 24 h.
binding affinity and selectivity for mGluR5. Important for
MPEP binding to mGluR5 are residues in TMIII and TMVII
on the one hand and TMV and TMVI on the other hand.21
Binding to these TMs represents the general motif of many
mGluR5 antagonists. Structurally, mGluR5 ligands such as
MPEP and M-MPEP or radioligands like [18F]3-fluoro-
5-(pyridin-2-ylethynyl)benzonitrile22 ([18F]F-PEB, Figure 1, 3),
[18F]3-fluoro-5-((2-methylthiazol-4-yl)ethynyl)benzonitrile22
([18F]F-MTEB, Figure 1, 6), and[18F]-3-fluoro-5-(2-(2-(fluoro-
methyl)thiazol-4-yl)ethynyl)benzonitrile23,24 ([18F]SP203,
Figure 1, 7) consist of a heterocycle, most often a pyridine
moiety that is connected via an acetylene linker to a second
aromatic or heteroaromatic ring system. However, one of the
most successful mGluR5 PET tracers and the first for imaging
mGluR5 in humans, (E)-3-((6-methylpyridin-2-yl)ethynyl)-
cyclohex-2-enone-O-[11C]methyloxime([11C]ABP688, Figure 1,
4),25 slightly differs from this pattern. A cyclohexenoneoxime
moiety (instead of a phenyl ring or a heteroaromatic ring
system) is connected to a pyridine ring via the acetylene linker.
Although 4 ([11C]ABP688) is an excellent imaging agent, its
use is limited to PET centers with a cyclotron and radio-
chemistry facilities mainly because of the short physical half-
life of carbon-11. Consequently, there is a need for a fluorine-
18 labeled analogue of 4 with similar imaging properties. The
optimal physical half-life of fluorine-18 (t1/2 = 110 min)
permits the shipping of fluorine-18 labeled compounds over
large distances and thus their commercial use.
Recently, our group reported on [18F](E)-3-((6-fluoropyridin-
2-yl)ethynyl)cyclohex-2-enone-O-methyloxime26 ([18F]F-PEC-
MO, Figure 1, 5), the first fluorinated analogue of 4 bearing
fluorine-18 at the pyridine functionality. Radiotracer 5 exhibits
high in vitro binding affinity toward mGluR5 but shows rapid
defluorination in vivo. In an effort to develop more stable
fluorinated analogues, we designed and synthesized several new
candidates that are amenable to fluorine-18 labeling by a one-
step radiolabeling approach. Structure-activity relationship
(SAR) studies were carried out to elucidate the optimal position
of the fluorine atom. At first, the position of the nitrogen in the
pyridine ring relative to the acetylene linker was shifted. In
another approach, the methyl group at the oxime functionality
in 4 was replaced by a variety of oxygen containing side chains,
pyridines, and benzonitriles. Herein, we report on the syntheses
and the binding affinities of these novel fluorinated analogues
and present the fluorine-18 labeling of the most promising
candidate.
Chemistry
In Scheme 1 is shown the synthetic pathway leading to
compounds 10 and 11. Alkyne derivative 9 was obtained as
previously described.26 Palladium catalyzed Sonogashira cou-
pling27 of 9 to 3-bromopyridine (8a) or 4-bromopyridine (8b)
gave 10 or 11, respectively, in reasonable yields. Compounds
16-18, which are key intermediates for the preparation of a
large series of 4 analogues, were prepared according to the
reaction pathway outlined in Scheme 2. The reaction of ketone
12 with hydroxylamine hydrochloride yielded a mixture of
The introductionofa fluorineatom intoa moleculechanges
its structure and therefore affects properties such as lipophi-
licity, binding affinity, and chemical and metabolic stability.

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4011
Scheme 2. Synthetic Pathway toward Key Intermediates 16-18a
a Reagents and conditions: (a) NH2OH HCl, pyridine, rt, 18 h; (b) Pd(PPh3)4, CuI, Et3N, DMF, rt, 20 h.
3
Scheme 3. Synthetic Pathway toward Fluoropyridines (Series 2 Compounds) and Fluorobenzonitriles (Series 3 Compounds)a
a Reagents and conditions: (a) DMF, NaH, rt, 2 h.
trans- and cis-oximes that were easily separated by flash
column chromatography to give pure trans-13 and cis-14 in
59% and 9% yields, respectively. The Sonogashira coupling of
the (E)-3-ethinylcyclohex-2-enoneoxime (13) with 15a and 15b
gave key intermediates 16 and 17, respectively, while the
coupling of (Z)-3-ethinylcyclohex-2-enoneoxime (14) to 15a
afforded compound 18.
Target compounds 20-23 were obtained by the reaction of
2,6-difluoropyridine (19a) with oximes 16-18 (Scheme 3).
Synthon 16 was reacted with 2,3-difluoropyridine (19b) to
give exclusively compound 22. A third series of compounds
containing fluorobenzonitriles at the oxime functionality of
the 4 core structure is depicted in Scheme 3.
The synthesis of the compounds containing the cyano
group was accomplished by reacting oximes 16 and 18 with
2,4-difluorobenzonitrile (24). Starting material 24 has poten-
tially two positions for nucleophilic attack. Consequently,
four products 25-28 were obtained from the two syntheses.
Preferred was the substitution of the fluorine in para position
to the cyano group. As a result, compound 26 obtained as a
side product was not further characterized. Compound 25
could be used for further investigations. With oxime 16, two
products were obtained with 28 being the major product.
Series 4 compounds were obtained by the reaction of oxime
16 with oxygen containing aliphatic starting materials such as
29, 30, and 3328 as shown in Scheme 4. Compound 31 was
synthesized by reacting 16 with 1-fluoroethanol (29) and
dibromomethane in the presence of sodium hydride. The
synthesis gave the desired end product 31 in moderate yields.
Compound 32 was prepared in a similar way using 1-fluoro-
propanol (30) as starting material. For the synthesis of com-
pound 38 (Scheme 4), an analogue with longer side chain, a
slightly different approach was chosen starting from (2-(2-
bromoethoxy)ethoxy)(tert-butyl)dimethylsilane (33)28 which
was prepared according to literature procedure. The addition
of 33 to key intermediate 16 afforded 34 in good yield.
Compound 34 was converted to the corresponding tosylate
36 after cleavage of the TBS group and reaction with tosyl
chloride. Reaction of 36 with dry tetra-n-butylammonium
fluoride (TBAF) resulted in the final product 38. Since 38

4012 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Baumann et al.
Scheme 4. Synthetic Pathway toward 4 Analogues with Oxygen Containing Side Chains (Series 4 Compounds)a
a Reagents andconditions:(a)DMF, NaH,CH2Br2, 40min, rt;(b) (2-(2-bromoethoxy)ethoxy)(tert-butyl) dimethylsilane (33), DMF, NaH, 2h, rt; (c) (1)
THF, TBAF, 1.5 h, rt; (2) CH2Cl2, Et3N, benzenesulfonyl chloride, 0 °C, 8 h; (d) TBAF, THF, 60 °C, 5 h; (e) K[18F]F-Kryptofix 222, DMF, 90 °C, 10 min.
exhibited high binding affinity to mGluR5, compound 39
derived from key intermediate 17 was similarly synthesized for
direct comparison (Scheme 4). For the one-step radiosynth-
esis of compound [18F]38, tosylated synthon 36 served as the
precursor.
binding. The resulting inhibition constants (Ki) of the com-
pounds are listed in Table 1. The prototypical pyridine-3-yl
and pyridine-4-yl analogues 10 and 11 with Ki values of 252 (
25 and 54.8 ( 20.2 nM, respectively, were shown to be mod-
erately potent binders with clearly reduced binding affinity
compared to the lead compound 4 (Ki = 4.4 nM). The
strategy to alter the position of the pyridine N-atom to
stabilize analogues of 5 was thus not further pursued.
Results and Discussion
Chemistry. The total syntheses of all target compounds
were accomplished by convergent syntheses. The intermedi-
ates and products were obtained overall in satisfactory
yields, although none of the synthesis steps was optimized.
However, the formation of compounds 10 and 11 by Sono-
gashira coupling resulted in relatively low yields of 25 and
13%, respectively. In both cases purification by column
chromatography over silica was problematic. Flash column
chromatography finally turned out to be more efficient. The
addition of dibromomethane to the sodium salts of com-
pounds 16, 29, and 30 afforded compounds 31 and 32 in 31%
and 18% yields, respectively. The low yields are probably
due to competing reactions. But for our purposes, the yields
are quite acceptable.
Structure-Activity Relationship Studies. Thirteen novel
analogues of 4 were successfully synthesized and investigated
for their binding affinity toward mGluR5. Competitive bind-
ing experiments were carried out with each candidate using
[3H]M-MPEP, a well characterized mGluR5 ligand, and
employing rat brain membranes without cerebellum. Non-
radioactive 4 was used for the determination of nonspecific
We next explored the possibility of incorporating substi-
tutions at the oxime moiety of the core structure in order to
investigate the tolerability of these substituents. An extensive
survey of fluorine substituted pyridines and benzonitriles
with several trans- and cis-oxime isomers of 4 was conducted.
Also, derivatives of 4 lacking the methyl group in the pyri-
dine ring were synthesized. The goal of improving or retain-
ing nanomolar binding affinity was achieved for the series 2
compounds, in particular for compounds 21 (Ki = 9.15 (
2.15 nM) and 22 (Ki = 4.01 ( 0.97 nM), which are both
desmethyl analogues of 4. From these series, the 3-fluoro
substitution in the oxime-linked pyridine ring (R2) exhibited
the highest binding affinity. The corresponding methyl con-
taining compound 20, on the contrary, showed a 5-fold
reduced affinity, which is ascribed to the presence of the
methyl group on the acetylene-linked pyridine ring (R1).
However, other SAR studies showed that the presence of a
methyl group in ortho position to the N-atom in heterocycles
such as the pyridine ring (R1) in 4 (Table 1) is well tolerated
for mGluR5 binding or even improves mGluR5 binding.30-32
Thus, we hypothesized that the reduction in binding affinity

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4013
Table 1. In Vitro Binding Data and clogP/log DpH7.4 Values of Analo-
gues of 4a
size, the two bulky moieties of 20 may exceed the size of the
available space in the binding pocket. In terms of lipophilicity,
the increase in Ki of the methylated ligand 20 would follow
the general trend shown in Figure 3 whereby compounds with
Ki < 10 nM have generally clogP values between 1.7 and 2.5,
whereas compounds with Ki g 10 exhibited log P values
greater than 2.5. Both hypotheses are in agreement with the
Ki values of compounds 38 (Ki = 3.8 ( 0.4 nM) and 39 (Ki =
26.8 ( 7.1 nM). Also in this case, a 7-fold decreased affinity
could be observed for the methyl containing derivative 39.
The addition of relatively bulky R2 groups, i.e., pyridines
(compounds 20-23) or benzonitriles (compounds 25-27),
including compounds with cis orientation (compounds 23
and 25), affected the binding affinity of the derivatives of the
lead compound 4 to a significantly lower extent than the shift
of the nitrogen in compound 10. Nonetheless, these com-
pounds (20-23, 25-28) did not show high enough binding
affinity to be considered for further evaluation.
There were no significant differences in the binding affi-
nities of the cis- and trans-isomers of the benzonitriles
(compounds 25 and 27, Table 1), again suggesting that the
large size and/or the high lipophilicity of the benzonitrile
moiety rather than structural features of the ligands reduced
the binding affinity. In case of the smaller pyridine (R2)
analogues, Ki differed by a factor of 5 when cis- and trans-
isomers 23 and 21 are compared. It is noteworthy that for
compound 4 the trans-isomer is significantly a more potent
binder than the cis-isomer.25 The pegylated desmethyl ana-
logues (31, 32, and 38) showed nanomolar binding affinities
with the longer side chains exhibiting slightly higher affi-
nities. The position of the oxygen in the side chain had only a
marginal effect on the binding affinity.
The binding of MPEP and M-MPEP to mGluR5 involves
TMIII, TMVII, TMV, and TMVI.18 Whether 4 and our
new ligands bind to exactly the same domains and whether
they interact with similar amino acids or not remain unan-
swered. Nevertheless, the general structure of successful
mGluR5 ligands, as described by Kulkarni et al.,33 was
retained for every novel candidate. Often, two aromatic
moieties are connected via a convenient linker. This provides
for interaction with two hydrophobic binding areas within
the binding pocket of mGluR5. Keeping this concept led to
the development of compound 38. From all the analogues of
4 synthesized, compound 38 exhibited the highest binding
affinity (Ki = 3.8 ( 0.4 nM) to mGluR5 and was therefore
selected for further evaluation as a PET tracer candidate.
Radiosynthesis and Saturation Binding Studies with [18F]38.
For the most promising candidate 38, a radiosynthetic
procedure for its [18F]-labeled analogue, [18F]-38, was suc-
cessfully established (Scheme 4). The conversion of the
tosylate precursor 36 into [18F]38 was accomplished using
Kryptofix and K2CO3 as base in N,N-dimethylformamide
(DMF). The reaction proceeded for 10 min at 90 °C and gave
radiochemical yields between 25% and 35% (decay corrected).
Semipreparative HPLC purification using acetonitrile (MeCN)
and water as mobile phase resulted in high radiochemical
purity (g96%) of the final product. The specific activity of
[18F]38 was in the range 110 - 350 GBq/μmol at the time of
quality control.
a The Greek symbols in the table have the following meaning: (R) Ki
data obtained from three independent competition binding experiments
in triplicate with the radioligand [3H]M-MPEP and rat brain homo-
genate containing mGluR5; data are presented as the mean value
followed by the standard deviation; (β) Ki data obtained from two
independent competition binding experiments in triplicate; (γ) Ki data
determined according to the standard procedure for competition bind-
ing experiments as a single determination in triplicate; (δ) calculated
log P values (ChemDraw); (ε) experimentally obtained values (n = 5).
The shake-flask method34 was used to determine the log
DpH7.4 of [18F]38. As expected from clogP calculations
(Table 1), this ligand displays a moderate lipophilicity with
a log D value of 1.7 ( 0.1 (Table 1, 38). The measured
log DpH7.4 suggests that [18F]38 is sufficiently lipophilic for
was related either to the overall size of the methylated
analogue 20 or to the increased lipophilicity. Regarding the

4014 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Baumann et al.
Figure 2. (A) Typical saturation binding curve of [18F]38 binding to rat brain membranes. (B) Representative Scatchard plot of [18F]38
saturation data. The dissociation constants obtained from three independent experiments are KD1 = 0.6 ( 0.2 nM and KD2 = 13.7 ( 4.7 nM.
free diffusion across the blood-brain barrier,35 although this
value is somewhat lower than the experimentally determined
log D value of 4. Efforts to increase the lipophilicity, however,
resulted in reduced affinity as shown in Figure 3.
Further characterization of [18F]38 in a saturation binding
assay with rat brain membranes revealed excellent binding
affinity. A typical saturation curve and Scatchard plot analysis
are shown in Figure 2. The Scatchard plots of three indepen-
dent experiments were not linear as would be expected for one
or more binding sites with equal binding affinity but were
characterized by a steeper slope in the low concentration range
than in the high concentration range. This was independent of
whether total or specific binding was used for the analysis (data
not shown). The binding data were fitted with a model assum-
ing two different binding sites. The respective binding para-
meters for [18F]38 are KD1 = 0.6 ( 0.2 and KD2 = 13.7 ( 4.7
nM and Bmax1 and Bmax2 values ranging from 470 to 1870 fmol/
mg and from 4.0 to 15.6 pmol/mg protein, respectively. On the
basis of these data, we cannot conclude whether both binding
sites are on mGluR5 or not. Note that the number of accessible
binding sites is about 8 times higher for the site with the weaker
binding affinity. The nonlinear Scatchard plots could alterna-
tively indicate varying affinity states of one single binding site
because of different receptor states, receptor-effector cou-
pling, or decreasing receptor affinity caused by increasing
receptor occupancy. Finally, artifacts due to technical problems
are possible reasons for nonlinear Scatchard plots.36
Figure 3. Correlation between inhibition constant (Ki) and clogP
values of ligands 20-39.
3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB),39
binding to the binding site of negative allosteric modulators of
mGluR5 such as MPEP in parallel. As Chen et al. hypothe-
size, CDPPB acts at overlapping binding sites in the trans-
membrane domain of mGluR5. However, the existence of
multiple binding sites could lead to the results described above
for compound 38. Further studies are required to characterize
the mGluR5 binding of 38 in more detail and to exclude high-
affinity binding to other structures. Target specificity is indis-
pensable for a successful PET tracer.
So far, no mGluR5 antagonist sharing the same binding
site as M-MPEP has been reported to bind to two distinct
binding sites at the mGluR5. Only recently, Chen et al.37,38
observed for a positive allosteric modulator of mGluR5,
Conclusion
The synthesis and characterization of 13 novel analogues of
4 were successfully carried out. The binding affinities of the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4015
compounds show that substituents at the oxime functionality
are well tolerated. Five compounds exhibited Ki values below
10 nM. The most promising compound 38 was successfully
labeled with fluorine-18 using a single-step radiosynthetic
approach. The high binding affinity of [18F]38 to mGluR5
was confirmed by in vitro tests. Further in vitro evaluation
and preclinical in vivo imaging with [18F]38 will show whether
this tracer can even be used as a PET imaging agent for
mGluR5 in humans.
Total binding of radioligand was obtained with only buffer and
membranes. The test samples with a total volume of 200 μL were
incubated for 45 min at room temperature (rt). In order to separate
free radioligand from the membranes, 4 mL of ice-cold incubation
buffer II was added followed by vacuum filtration over GF/C filters
(Whatman). After rinsing the filters twice with 4 mL of buffer II,
4 mL of scintillation liquid (Ultima Gold, Perkin-Elmer) was added
to the filters in β scintillation vials. With a β counter (Beckman
Instruments, LS 6500, multipurpose scintillation counter) the
radioactivity retained on the filters was measured. The data were
evaluated with KELL Radlig software (Biosoft). For calculation of
the Ki the Cheng-Prusoff equation is the underlying equation.
Three independent experiments were performed for each end
compound, except for compounds 11 and 22 (n = 2).
Experimental Section
General Methods. Reagents and solvents utilized for experi-
ments were obtained from commercial suppliers (Sigma-Aldrich,
Alfa Aesar, Merck, and Fluka) and were used without further
purification unless stated otherwise. [3H]M-MPEP was provided
byNovartis. Thinlayer chromatography performedonprecoated
silica gel 60 F245 aluminum 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 following abbreviations
are used for the description of 1H NMR and 13C NMR spectra:
singlet (s), doublets (d), triplet (t), quartet (q), quintet (quint),
doublet of doublets (dd), multiplet (m). The chemical shifts of
complex multiplets are given as the range of their occurrence.
Low resolution mass spectra (LRMS) were recorded with a
Micromass Quattro micro API LC electrospray ionization
(ESI). High resolution mass spectra (HRMS) were recorded with
a Bruker FTMS 4.7 T BioAPEXII (ESI). Quality control of the
final products in order to confirm the purity ofg95% of the novel
compounds was achieved by analytical high performance liquid
chromatography (HPLC). It was performed on an Agilent 1100
system equipped with a radiodetector from Raytest using a
reversed phase column (Gemini 10 μm C18, 300 mm ꢀ 3.9 mm,
Phenomenex) by applying an isocratic solvent system with 70%
MeCN in water and a flow of 1 mL/min.
Membrane Preparation. Competition binding assays for deter-
mination of mGluR5 binding affinity were carried out using rat
brain membranes. For the preparation of these membranes male
Sprague-Dawley rats were sacrificed by decapitation followed
by quick removal of the brain including the olfactory bulb. After
separation of the cerebellum the brain tissue 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, Kine-
matica AG) for 1 min at setting 4. The obtained homogenate
was centrifuged (1000g, 15 min, 4 °C) to give a pellet (P1). The
supernatant was collected, and P1 was resuspended in 5 volumes
of sucrose buffer. After homogenization and centrifugation, the
supernatant was collected and combined with the supernatant
before. Centrifugation of the obtained mixture (17000g, 20 min,
4 °C) resulted in a pellet (P2), which was resuspended with incu-
bation buffer (5 mM Tris/acetate buffer, pH 7.4). The suspension
was again centrifuged (17000g, 20 min, 4 °C) followed by resus-
pension of the resulting pellet with incubation buffer to obtain the
final membrane preparation suitable for storage at -70 °C. For
assays, the membranes were thawed and kept on ice during all of
the preparation processes. The protein concentration was deter-
mined before each experiment by Bio-Rad microassay with
bovine serum albumin as a standard (Bradford).40
Saturation Assay. P2 membranes (500 μg/mL) were incubated
with increasing concentrations of [18F]38 (0.25-100 nM) each in
triplicate in ice-cold incubation buffer II to give a total volume
of 200 μL. Nonspecific binding was determined in the presence
of 100 μM unlabeled 4 for each concentration of [18F]38 in
triplicate. The test samples were incubated for 45 min at rt before
addition of 4 mL of ice-cold incubation buffer II, and vacuum
filtration over GF/C filters (Whatman) pretreated with 0.05%
PEI solution was performed. The filters were rinsed twice with
4 mL of incubation buffer II before each filter was prepared for
measurement in an appropriate vial for measurement of the
activity retained on the filter using a γ-counter (Wizard, Perkin-
Elmer). Data analysis was performed for saturation and Scatch-
ard analysis with Kell-Radlig computer program (McPherson &
Biosoft, Cambridge, U.K.,1997). Three independentexperiments
were carried out with [18F]38 obtained from three independent
radiosynthetical productions.
Determination of the Lipophilicity of [18F]38 and of [18F]39
(log DpH7.4). Radiosynthesis of [18F]39 was performed in ana-
logy to the radiolabeling for [18F]38 (see below). As described in
Wilson et al.34 for the shake flask method, 500 μL of octanol
saturated with phosphate buffer (Soerensen, pH 7.4) and 500 μL
of phosphate buffer saturated with octanol were pipetted into an
Eppendorf cup. After addition of 10 μL of radiotracer solution
the samples were shaken for 15 min. Centrifugation at 5000 rpm
for 3 min was performed to separate the phases. Then 50 μL of
each phase was pipetted into Eppendorf cups for measurement
of the distribution of the activity with a γ-counter (Wizard,
Perkin-Elmer). The experiment was performed in quintuplicate.
Radiosynthesis of [18F]38. [18F]Fluoride was produced via the
18O(p,n)18F reaction in a cyclotron using enriched 18O-water.
For trapping of 18F- from the aqueous solution it was passed
through a light QMA cartridge (Waters) preconditioned with
0.5 M K2CO3 (5 mL) and water (5 mL). For elution of 18F- from
the cartridge into a tightly closed 5 mL reaction vial, 1 mL of
Kryptofix K222 solution (Kryptofix K222; 2.5 mg of K2CO3,
0.5 mg in MeCN/water (3:1)) was used. The solvents were
evaporated at 110 °C under vacuum in the presence of a slight
in-flow of nitrogen gas. After addition of MeCN (1 mL), azeo-
tropic drying was carried out as described above. The drying
procedure was repeated twice. A solution of the corresponding
precursor 36, 2 mg in 300 μL of dry DMF, was then added to the
kryptofix complex and the reaction mixture was heated at 90 °C
for 10 min before 2 mL of MeCN in water (1:1) was added.
Purification by semipreparative HPLC was carried out on a
HPLC system equipped with a Merck-Hitachi L-6200A intelli-
gent pump, a Knauer variable wavelength monitor UV detector,
€
and a Geiger Muller LND 714 counter with Eberlein RM-14
instrument using a reversed phase column (Gemini 5 μm C18,
250 mm ꢀ 10 mm, Phenomenex) with a solvent system and
gradient as follows: H2O (solvent A), MeCN (solvent B); flow
5 mL/min; 0-10 min, 5% B; 10-20 min, 5% B f 50% B; 20-50
min, 50% B. The fraction containing the product was collected,
andthe mobilephasewas evaporatedundervacuum. The product
was finally dissolved in PEG200/water (1:1). Determination of
relative lipophilicity, radiochemical purity, and specific activity
Competition Binding Assays. P2 membranes were incubated
with increasing concentrations of test compound (1 pM to 100 μM),
each in triplicate, in incubation buffer II (30 mM 4-(2-hydro-
xyethyl)piperazine-1-ethanesulfonic acid sodium salt (HEPES),
110 mM NaCl, 5 mM KCl, 2.5 mM CaCl2 H20, 1.2 mM MgCl2,
pH 8). Radioligand [3H]M-MPEP (2 nM) was added. Nonspecific
3
binding was determined in the presence of unlabeled 4 (100 μM).

4016 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Baumann et al.
was carried out during the quality control step. Identification of
[18F]38 was achieved by coinjection with reference 38 to the
HPLC system during quality control.
Finally, benzenesulfonyl chloride (114.39 mg, 0.6 mmol) was
added, and after 8 h the mixture was diluted with water. Extrac-
tion with Et2O (3 ꢀ 20 mL) and washing the organic layers with
water (2 ꢀ 20 mL) and brine (30 mL) before drying over Na2SO4
led after evaporation to 176 mg of crude product. Purification by
column chromatography using Et2O/pentane (5:1) resulted in a
yellow oil (67 mg, yield = 50%). 1H NMR (400 MHz, CDCl3):
δ 8.59 (d, J = 5.2 Hz, 1H), 7.80 (d, J = 8.4 Hz, 2H), 7.65 (t, J =
8.0 Hz, 1H), 7.44 (d, J = 8.1 Hz, 1H), 7.33 (d, J = 8.3 Hz, 2H),
7.22 (t, J = 6.6 Hz, 1H), 6.56 (s, 1H), 4.17 (q, J = 4.2 Hz, 4H),
3.69 (q, J = 4.5 Hz, 4H), 2.55 (t, J = 7.1 Hz, 2H), 2.44 (s, 3H),
2.40 (t, J = 6.1 Hz, 2H), 1.80 (quint, J = 6.4 Hz, 2H). 13C NMR
(100 MHz, CDCl3): δ 20.8, 21.7, 22.6, 29.4, 68.7, 69.2, 69.8, 77.2,
89.8, 91.9, 122.9, 127.3, 127.4, 128.0, 129.8, 130.9, 133.0, 136.2,
143.2, 144.8, 150.1, 155.8. MS m/z 455.01 (Mþ). HRMS calcd for
C24H27N2O5Sþ, 455.1635; found, 455.1631.
(E)-3-(Pyridin-2-ylethynyl)cyclohex-2-enoneoxime (16). A solu-
tion of 2-bromopyridine 15a (2.444 g, 15.5 mmol) in DMF (12 mL)
was carefully degassed and placed under argon atmosphere before
Pd(PPh3)4 (110 mg, 0.097 mmol) was added. After the mixture was
stirred for 5 min, Et3N (6 mL) was added, and after further 5 min
CuI (77 mg, 0.4 mmol) was added. A solution of 13 (2.013 g, 14.9
mmol) in DMF (20 mL) was prepared and poured into the
reaction mixture. After stirring the mixture for 24 h at rt the
synthesis was quenched with saturated NH4Cl and extracted with
EtOAc. The combined organic layers were washed with water and
brine before evaporation under reduced pressure. Purification by
column chromatography pentane/EtOAc (85:15 f 7:3) led to
yellow crystals (2.7 g, yield = 85%). 1H NMR (400 MHz, CDCl3):
δ 8.59 (d, J = 5.2 Hz, 1H), 7.67 (t, J = 7.8 Hz, 1H), 7.46 (d, J =
8.3 Hz, 1H), 7.23 (t, J = 6.2 Hz, 1H), 6.63 (s, 1H), 2.64 (t, J =
6.8 Hz, 2H), 2.41 (t, J = 6.8 Hz, 2H), 1.83 (quint, J = 6.8 Hz, 2H).
13C NMR (100 MHz, CDCl3): δ 20.8, 21.5, 29.2, 90.3, 91.5,
122.9, 127.0, 127.2, 131.8, 136.4, 143.1, 149.9, 156.2. MS m/z
212.95 (M þ H)þ. HRMS calcd for C13H12N2O, 212.0950;
found 212.0945.
(E)-3-(Pyridin-2-ylethynyl)cyclohex-2-enone-O-2-(2-(tert-butyl-
dimethylsilyloxy)ethoxy)ethyloxime (34). To a solution of trans-
3[(pyridine-2-yl)ethynyl]cyclohex-2-enoneoxime 16 (151 mg, 0.71
mmol) inDMF(15 mL) wasadded60%NaH(37mg, 0.92mmol).
After the mixture was stirred for 30 min at rt, 2-(2-bromoethoxy)-
(tert-butyl)dimethylsilane 33 (262 mg, 0.92 mmol) was added
while the color changed quickly from yellow to light brown. After
1.5 h, 50% NaHCO3 solution was added, and after a further 5 min
the reaction mixture was extracted with ethyl acetate (3 ꢀ 20 mL).
The combined organic layers were washed with water (2 ꢀ 20 mL)
and brine and dried over Na2SO4 before the solvent was evapo-
rated under reduced pressure. The obtained crude product was
purified by column chromatography over silica using pentane/
EtOAc (6:4), which gave a yellow oil (253 mg, yield = 86%). 1H
NMR (400 MHz, CDCl3): δ 8.59 (d, J = 5.9 Hz, 1H), 7.65 (t, J =
7.4 Hz, 1H), 7.44 (d, J =8.2 Hz, 1H), 7.22 (t, J = 6.4Hz, 1H), 6.57
(s, 1H), 4.26 (t, J = 5.2 Hz, 2H), 3.76 (q, J = 5.1 Hz, 4H), 3.56 (t,
J = 5.3 Hz, 2H), 2.58 (t, J = 6.8 Hz, 2H), 2.40 (t, J = 6.1 Hz, 2H),
1.80 (quint, J = 6.9 Hz, 2H), 0.90 (s, 9H), 0.07 (s, 6H). 13C NMR
(100 MHz, CDCl3): δ -5.3, 18.4, 20.8, 22.3, 25.9, 29.4, 62.8, 69.8,
72.7, 73.8, 90.1, 91.6, 122.9, 127.1, 127.3, 131.2, 136.3, 143.1, 150.0,
155.6. MS m/z 415.09 (M þ H)þ. HRMS calcd for C23H34N2-
NaO3Siþ, 437.2231; found, 437.2224.
(E)-3-((6-Methylpyridin-2-yl)ethynyl)cyclohex-2-enone-O-
2-(2-(tert-butyldimethylsilyloxy)ethoxy)ethyloxime (35). This com-
pound was prepared in an analogous way to 34. Starting materials
17 (491 mg, 2.17 mmol) in DMF (40 mL) and 33 (550 mg, 1.94
mmol) gave the desired product (380 mg, yield = 45%). 1H NMR
(400 MHz, CDCl3): δ 7.53 (t, J = 7.9 Hz, 1H), 7.27 (d, J = 7.4 Hz,
1H), 7.08 (d, J = 7.2 Hz, 1H), 6.56 (s, 1H), 4.25 (t, J= 5.5 Hz, 2H),
3.78-3.73 (m, 4H), 3.58 (t, J = 5.2 Hz, 2H), 2.58 - 2.55 (m, 5H),
2.38 (t, J = 7.1 Hz, 2H), 1.78 (quint, J = 6.8 Hz, 2H), 0.89 (s, 9H),
0.06 (s, 6H). 13C NMR (100 MHz, CDCl3):δ-5.2, 18.4, 20.8, 22.3,
26.0, 29.4, 62.8, 69.8, 72.7, 73.4, 73.8, 89.5, 92.9, 122.7, 124.5, 127.3,
131.0, 136.4, 142.7, 156.0, 159.2. MS m/z: 429.16 (M þ H)þ.
(E)-2-(2-(3-(Pyridin-2-ylethynyl)cyclohex-2-enylideneaminooxy)-
ethoxy)ethyl 4-Methylbenzenesulfonate (36). To a solution of 34
(230 mg, 0.55 mmol) in dry tetrahydrofuran (THF, 12.5 mL) was
added 1 M TBAF (195 μL). After the mixture was stirred for for
1 h, water (10 mL) was added. The solution was extracted with
EtOAc (3 ꢀ 20 mL), and the organic layers were washed with
water (2 ꢀ 20 mL) and brine (30 mL) before drying over Na2SO4.
The brown crude was obtained after evaporation of the solvent
under reduced pressure. It was used without further purification.
3-[Pyridine-2-yl)ethynyl]cyclohex-2-enone-O-(2-oxyethyl)oxy-
ethyloxime (crude, 85 mg) was dissolved in dry CH2Cl2 (2 mL).
After addition of Et3N (118 μL) the mixture was set to 0 °C.
3-[Pyridine-2-yl)ethynyl]cyclohex-2-enone-O-fluoroethyloxy-
ethyloxime (38). TBAF 3H2O (227 mg, 0.71 mmol) was dried on
3
a high vacuum system at 45 °C for 24 h and was then dissolved in
dry THF (5 mL). A solution of 36 (130 mg, 0.29 mmol) in dry
THF (12 mL) was added, and after stirring the mixture for 5 h at
60 °C, water (10 mL) was added. The solution was extracted with
Et2O, and the organic layers were washed with water (2 ꢀ 20 mL)
and brine (1 ꢀ 20 mL) before drying over Na2SO4. The brown
crude, which was obtained after evaporation, was purified by
column chromatography over silica with Et2O/pentane (2:1)
which afforded the desired product (49 mg, yield = 56%), 95%
purity by HPLC (tR = 4.98 min). 1H NMR (400 MHz, CDCl3):
δ 8.59 (d, J = 5.0 Hz, 1H), 7.67 (t, J = 7.8 Hz, 1H), 7.44 (d, J =
8.1 Hz, 1H), 7.22 (t, J= 6.9 Hz, 1H), 6.57 (s, 1H), 4.63 (t, J=4.2Hz,
1H), 4.51 (t, J = 4.2 Hz, 1H), 4.28 (t, J = 5.1 Hz, 2H), 3.79 (m,
3H), 3.71 (t, J = 4.0 Hz, 1H), 2.58 (t, J = 6.4 Hz, 2H), 2.40 (t, J =
6.2 Hz, 2H), 1.80 (quint, J = 6.3 Hz, 2H). 13C NMR (100 MHz,
CDCl3): δ 20.8, 22.3, 24.6, 70.2, 70.7 (d, J = 19.1 Hz), 73.4, 83.5
(d, J = 168.0 Hz), 90.2, 92.2, 123.2, 127.6, 127.7, 131.4, 136.5,
143.6, 150.5, 156.1. 19F NMR (376 MHz, CDCl3): δ -223.10 (m,
J = 20.36 Hz). MS m/z 303.01 (Mþ). HRMS calcd for C17H20-
FN2O2, 303.1503; found, 303.1498.
Acknowledgment. We acknowledge the technical support
of Claudia Keller, Mathias Nobst, Lukas Dialer, and Phoebe
Lam. We thank Christophe Lucatelli and Stefanie-Dorothea
€
Kramer for fruitful discussions.
Supporting Information Available: Synthesis procedures,
NMR spectroscopic data, MS analytical data for intermediates
13, 14, 17, 18, and 34-37 and final compounds 10, 11, 20-28,
31, 32, and 39, and HPLC results for final compounds 10, 11,
20-28, 31, 32, and 39. This material is available free of charge
via the Internet at http://pubs.acs.org.
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