Synthesis and evaluation of novel α-fluorinated (E)-3-((6- methylpyridin-2-yl)ethynyl)cyclohex-2-enone-O-methyl oxime (ABP688) derivatives as metabotropic glutamate receptor subtype 5 PET radiotracers

Article abstract of DOI:10.1021/jm300648b

In the search for an optimal fluorine-18-labeled positron emission tomography (PET) radiotracer for imaging metabotropic glutamate receptor subtype 5 (mGluR5), we have prepared a series of five α-fluorinated derivatives based on the ABP688 structural mani

Full text of DOI:10.1021/jm300648b

Article
pubs.acs.org/jmc
Synthesis and Evaluation of Novel α‑Fluorinated (E)‑3-((6-
Methylpyridin-2-yl)ethynyl)cyclohex-2-enone‑O‑methyl Oxime
(ABP688) Derivatives as Metabotropic Glutamate Receptor Subtype
5 PET Radiotracers
Selena Milicevic Sephton,† Linjing Mu,† W. Bernd Schweizer,‡ Roger Schibli,† Stefanie D. Kramer,†
̈
and Simon M. Ametamey*,†
†Center for Radiopharmaceutical Sciences of ETH, PSI and USZ, Department of Chemistry and Applied Biosciences, Swiss Federal
Institute of Technology (ETH) Zurich, Wolfgang-Pauli Strasse 10, 8093, Zurich, Switzerland
‡Laboratory of Organic Chemistry, Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH)
Zurich, Wolfgang-Pauli Strasse 10, 8093 Zurich, Switzerland
S
* Supporting Information
ABSTRACT: In the search for an optimal fluorine-18-labeled
positron emission tomography (PET) radiotracer for imaging
metabotropic glutamate receptor subtype 5 (mGluR5), we have
prepared a series of five α-fluorinated derivatives based on the
ABP688 structural manifold by application of a two-step enolization/
NFSI α-fluorination method. Their binding affinities were evaluated
in vitro, and the most promising candidate (Z)-16 exhibited a Ki of
5.7 nM and a clogP value of 2.3. The synthesis of the precursor
tosylate (E)-22 revealed a preference for the (E)-configurational
isomer (Ki = 31.2 nM), and successful radiosynthesis afforded (E)-
[18F]-16 which was used as a model PET tracer to establish plasma
and PBS stability. (E)-[18F]-16 (Kd = 70 nM) exhibited excellent
specificity for mGluR5 in autoradiographic studies on horizontal rat brain slices in vitro.
INTRODUCTION
■
Positron emission tomography (PET) is a noninvasive imaging
technique in which 3D concentration images are obtained
through computational analysis of pairs of γ rays emitted
indirectly from compounds containing positron emitting
nuclides such as [11C] or [18F].1−3 Metabotropic glutamate
receptor subtype 5 (mGluR5) is a G-protein-coupled
postsynaptic receptor, and it belongs to group I of
metabotropic glutamate receptors, which together with
ionotropic glutamate receptors regulate glutamate, a major
excitatory neurotransmitter in mammalian brain.4−8 In 2006,
the Ametamey group reported on the synthesis, radiolabeling,
and pharmacological evaluation of [11C]-1 ([11C]-ABP688,
Figure 1) and subsequently illustrated its application as a PET
radiotracer for imaging of mGluR5 in vivo in human
subjects.9−11 The success of this first mGluR5 PET tracer
was immediate, and [11C]-1 was employed in many clinical
studies12−19 particularly because mGluR5 emerged as an
important drug target due to its demonstrated involvement in
long-term potentiation processes as well as several CNS
disorders20 (e.g., schizophrenia,21 depression,22 neuropathic
pain,23,24 drug addiction,25 Fragile X syndrome,26 and
Alzheimer’s19,27 and Parkinson’s disease28,29). Although clin-
ically applied with success, [11C]-1 has one significant limitation
Figure 1. Structures of carbon-11 and fluorine-18 mGluR5 PET
radiotracers from the Ametamey group and the synthetic plan to a new
series of α-fluorinated analogues of 1. A crossed double bond is used
to indicate double bond isomers (E and Z).
which is the short physical half-life (20 min) of the carbon-11
nuclide that limits its application to facilities with an on-site
cyclotron. This opened the possibility for further advancement
of mGluR5 PET tracers with the aim of designing a fluorine-18-
labeled tracer.
Received: May 9, 2012
Published: July 23, 2012
© 2012 American Chemical Society
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Fluorine-18 has a longer physical half-life (110 min) and, to
date, two of the most successful mGluR5 18F-labeled PET
tracers are 3-fluoro-5-(2-([18F](fluoromethyl)thiazol-4-yl)-
ethynyl)benzonitrile ([18F]-SP203), developed by the Pike
group,30−32 which showed limited defluorination in human
subjects, and 3-[18F]fluoro-5-(2-pyridinylethynyl)benzonitrile
([18F]-FPEB), developed by the Hamill group33,34 which has
also been applied in human subjects but is typically obtained in
low radiochemical yields. Neither Pike’s nor Hamill’s analogues
were based on the structural scaffold of 1, and the success of
[11C]-1 in human clinical practice prompted us to explore close
analogues of [11C]-1. Among other evaluated fluorinated
radiotracers35−39 to date, (E)-3-(pyridin-2-ylethynyl)cyclohex-
2-enone O-(2-(2-[18F]fluoroethoxy)ethyl) oxime ([18F]-
FDEGPECO, Figure 1)40,41 was identified as the most
promising candidate, which was successful in visualizing
mGluR5 in vivo in a rat brain without defluorination, albeit
with relatively high background activity.
Scheme 1. Optimization of α-Fluorination of Enone 6
Taking advantage of the success of [11C]-1 while
incorporating fluorine-18 with the more desirable physical
half-life, we aimed to investigate structures most closely related
to [11C]-1, in which a hydrogen atom is replaced by fluorine.
Apart from increasing the physical half-life of the radiotracer,
incorporation of a fluorine atom also alters chemical and
physical properties of the molecule particularly with respect to
the lipophilicity of the molecule, mainly due to the electro-
negativity of the fluorine atom.42−44 Fluorine-containing
molecules typically show increased log D when compared to
their nonfluorinated analogues,45 and this is favorable for the
development of CNS tracers, as it typically allows greater
permeation of the blood−brain barrier. On the basis of the ease
of their synthetic accessibility as well as their optimal clogP
values such that they are in the range of that of [11C]-1 (2.4),
we designed a series of α-fluoro-substituted compounds (Figure
1) and herein we report on their chemical syntheses, structural
elucidation, and determination of binding affinities, as well as
the establishment of the model PET radiotracer and evaluation
of its in vitro properties.
giving rise to related products in low yields requiring long
reaction times.49 To our knowledge, γ-fluorination50 was the
only application of NFSI with unsaturated ketones in which
case enolization was achieved using triphenylborane.
Recently, the MacMillan group and others described
advanced applications of NFSI for asymmetric α-fluorination
of aldehydes51−53 and ketones54 in excellent yields and high
enantioselectivity. In our hands, the enantioselective α-
fluorination of 4 under MacMillan’s reaction conditions
produced ent-9 in 8% yield and 94% ee. Our initial
pharmacological evaluation, however, was performed with
racemic α-fluoro samples.
In an attempt to optimize α-fluorination we explored several
other possibilities, but a two-step enolization/α-fluorination
was the most successful. Interestingly, direct lithiation of ethoxy
enone 5 with LDA48 followed by α-fluorination with NFSI led
to an inseparable mixture of 2- and 4-fluoro derivatives, and it
was not further explored. On the other hand, enone 10, which
was accessed via the Sonogashira coupling reaction at 60 °C,
was successfully enolized with LDA to give the TMS-enol ether
in 60% conversion, but the reaction with NFSI failed to yield 11
under conditions analogous to those for the α-fluorination of 8
(Scheme 2). Similarly, under MacMillan’s reaction conditions,
product was not formed.
Next, α-fluoro enone 9 was reacted with methyl or ethyl
hydroxylamine to afford the corresponding oximes 12 and 13 in
a ratio of geometrical isomers E:Z of 1:2 (Scheme 2). These
stable geometrical isomers were separable via column
chromatography. The assignment of E and Z configuration of
the oxime bond for isomers of 12 and 13 was initially made
based on the 1H NMR comparison to the known structure of 1
using the following principle. The hydrogen atom geminal to
the fluorine atom (Hf, Figure 2a) is shifted downfield when
closer through space to the oxime ether oxygen atom, and a
hydrogen on the cyclohexenyl double bond (HCy) is shifted
upfield when closer through space to the nitrogen atom lone
pair and vice versa (Figure 2a). In a comprehensive study,
Denmark and co-workers reported a similar geometrical
assignment of oxime double bond based on strong anisotropic
deshielding by the oxime oxygen on the equatorial protons at
C2 or C6 positions.55 Preference for the Z-isomer in this case
was attributed to additional anomeric stabilization of the sp2
RESULTS AND DISCUSSION
■
The synthesis of α-fluoro [11C]-1 derivatives was envisioned via
the Sonogashira cross-coupling of α-fluoro oxime ether 3 and
corresponding bromopyridines (2) as depicted in Figure 1.
Ethylene enone 6 (Scheme 1) was prepared by reacting
commercially available ethoxy enone 5 with ethynylmagnesium
bromide. In order to obtain α-fluoroenone 7, enone 6 was
treated with SelectFluor following the procedure reported by
Zupan and co-workers.46,47 While in methanol 6 was
completely unreactive, in acetonitrile a 19% conversion was
determined by NMR (entry 1 vs entry 2, Scheme 1). With
longer reaction times, conversion to 7 was significantly
improved (entries 2−4); however, purification by chromatog-
raphy on a silica gel column afforded only 8% of α-fluoroenone
7.
Alternatively, α-fluorination was accomplished via a two-step
procedure. In the first step, enone 6 was enolized with LDA
and quenched with chlorotrimethylsilane48 to afford TMS-enol
ether 8 with the concurrent protection of the free acetylene in
6. Silyl enol ether 8 was then fluorinated using N-
fluorobenzenesulfonimide (NFSI) in 24% yield, which could
not be improved regardless of the duration or temperature of
the reaction. Application of a two-step protocol enolization/
NFSI α-fluorination has been accomplished with ketones,
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lone pair on the nitrogen by the σ* bond between carbon
atoms at C1 and C2 positions in (Z)-TS-13 (Figure 2b).
With oximes 12 and 13 in hand, deprotection of TMS-
acetylene with TBAF was followed by the Sonogashira coupling
to give rise to α-fluoro derivatives 14 to 17 in good yields
(Table 1).
Scheme 2. Sonogashira Cross-Coupling Reaction To Form
α-Fluoro Derivatives of [11C]-1
Crystallization of (Z)-17 from Et2O allowed for X-ray crystal
structure analysis and confirmed the tentative configurational
assignment of the oxime ether double bond. Notably, (Z)-17
formed conglomerates, which could potentially be collected via
triage to obtain each enantiomer (Figure 3).56−58 X-ray crystal
Figure 3. ORTEP diagram for (Z)-17 showing Z-configuration of the
ethyl oxime ether double bond. Fifty percent probability ellipsoids are
plotted for non-hydrogen atoms.
structure analysis revealed a preferred conformation with the
fluorine atom in (Z)-17 in the axial position, as anticipated
from the geminal and vicinal coupling constants for Hf proton
(47.3 and 2.6 Hz, respectively). This finding is in agreement
with empirical observation made by the Stothers group for
conformationally constrained α-fluoro cyclohexanones.59
The biological evaluation of five derivatives 14−17 involved
the binding affinity (Ki) determination (Table 1). For this
purpose, a displacement assay with rat brain membranes was
employed, and the IC50 displacement curves for all five
derivatives are depicted in Chart 1. Structurally, the most
similar analogue to 1 is methyl oxime ether (Z)-15, which
showed reduced binding affinity in comparison to other
investigated oxime ethers. Out of the five analogues, the two
with the lowest Ki values, (Z)-14 and (Z)-16, were further
analyzed. Methyl oxime ether (Z)-14 exhibited the highest
binding affinity (3.4 nM), but the clogP and logD7.4 values
revealed low lipophilicity (1.7) of (Z)-14, which is below the
optimal range for a brain mGluR5 PET tracer.40 (Lipophilicity
values provided in Table 1 were used solely as guidelines to
establish relative relationship among investigated derivatives.
Predicted (clogP) using in silico method and experimentally
estimated (log D7.4) using HPLC method values showed
1
Figure 2. (a) H NMR analysis of E- and Z-isomers of 13. (b) The
anomeric stabilization rationale for a transition state (TS) leading to Z-
isomer.
Table 1. Product Yields of Sonogashira Cross-Coupling Reaction, Calculated (clogP), and Experimentally Estimated (log D7.4)
a
Lipophilicity Values and Related Ki Values As Determined via the Displacement Assay with [3H]-1
estimated
entry
code
R1
R2
oxime geometry
coupling yield (%)
clogP
log D7.4
Ki (nM)
1
2
3
4
5
6
7
14
15
16
16
17
1
Me
Me
Et
H
Me
H
Z
Z
Z
E
Z
E
Z
63
68
46
37
61
−
1.7
2.2
2.3
2.3
2.8
2.4
2.4
1.7
2.0
2.1
3.4 0.7
8.5 1.4
5.7 2.2
31.2 2.4
13.0 6.7
4.440
b
Et
H
2.1/3.5
2.5
Et
Me
Me
Me
Me
Me
2.3
9
1
−
1.9
−
a
b
Ki values are estimated from measured IC50 values using the Cheng−Prusoff equation (see Supporting information). Value calculated using the
HPLC method/value determined using the shake flask method. Note: Entries 6 and 7 are nonfluorinated analogues.
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Chart 1. [3H]-1 Displacement Curves for Derivatives 14−
excellent agreement.) We therefore decided to evaluate ethyl
oxime ether (Z)-16 which not only had a desired Ki value of 5.7
nM but also had a clogP of 2.3. Interestingly, this SAR study
revealed that the most significant difference in Ki was induced
by the configuration of the oxime ether double bond (E- vs Z-
16), similar to ABP688 oxime ether double bond isomers,9 and
minimal change in Ki binding affinity is observed with variation
of either the oxime ether (R1, Figure 1) or the pyridine ring
(R2, Figure 1) functionalities.
a
17
It was then desired to introduce fluorine-18 into the
molecule, but the application of an α-fluorination method
using [18F]-NFSI60 required an electrophilic source of fluorine-
18 (18F2), and this was not feasible for technical reasons. This
was due to the nucleophilic nature (18F−) of the fluorine-18
source produced in the cyclotron in our facilities. An alternative
approach was employed based on a nucleophilic substitution at
the α-position to the oxime ether, which required the
introduction of a suitable leaving group.
The tosyl leaving group was selected and the synthesis of
radiolabeling precursor 22 (Scheme 3) was envisioned via a
Rubottom oxidation61−63 of the previously prepared TMS-enol
ether 8.
Scheme 3. Synthesis of Radiolabelling Precursor Tosylate
(E)-22 via the Rubottom Oxidation
Trimethylsilyl enol ether 8 was treated with mCPBA64 to
afford epoxy enone 18 (Scheme 3). Enone 18 was purified only
to remove mCPBA residue on a silica plug, and as such it was
used for the next step in which the epoxide was opened under
basic conditions using a TBAF·THF complex to give rise to α-
hydroxy enone 19 in modest yields. α-Hydroxy enone 19 was
further converted to ethyl oxime ether 20 by reaction with O-
ethylhydroxyl amine in 64% yield. To our surprise, the Z:E ratio
of the oxime ether double bond was 1:10. Tentative assignment
of oxime ether double bond geometry was made based on the
γ-effect in the 13C NMR spectra65,66 of the two isomers (Figure
4a). In the two alkene molecules with all other identical
components, the sterically compressed carbon atom at the
allylic position is shifted upfield in comparison to the same
carbon with a higher degree of steric freedom. We applied the
γ-effect to ethyl oxime ethers (Z)-13 and (E)-13 to observe the
a
For each compound, three experiments were performed in triplicate,
and the average values are depicted in the curves (solid lines) fitted for
one binding site. Dotted lines represent estimated IC50 values.
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Scheme 4. Radiolabeling of Precursor Tosylate (E)-22 To
Afford (E)-[18F]-16 in 3% Decay-Corrected Radiochemical
Yield and 99% Purity As Determined by HPLC
radiochemical yield after purification via semipreparative HPLC
to afford model radiotracer (E)-[18F]-16 with 99% radio-
chemical purity. Initially performed at 90 °C for 10 min, a
decay-corrected radiochemical yield of 2% was obtained. A
slight improvement to 3% was accomplished by increasing the
reaction time to 13 min. The identity of the product was
confirmed through coinjection with cold reference (E)-16;
however, under the employed HPLC conditions for quality
control, E- and Z-isomers were inseparable. Due to similar
polarity of the two isomers, only partial separation was
accomplished using LiChrosorb column to show an E:Z ratio
of ca. 9:1, suggesting partial isomerization of the oxime ether
double bond during radiolabeling. A similar observation was
made for [11C]-ABP688 where typical E:Z ratio was ca. 10:1.9
The stability of the model compound (E)-[18F]-16 was
determined, and over a period of 2 h, PET radiotracer (E)-
[18F]-16 was stable when incubated at 37 °C in both rat plasma
and PBS. (Z)-Derivatives 14 to 17 are likely to exhibit similar
stability in plasma and PBS.
Figure 4. (a) Application of γ-effect for assignment of oxime ether
double bond. (b) Dipole−dipole minimization rationale for the
preference of E-isomer for α-hydroxy derivatives.
Lipophilicity of (E)-[18F]-16 was determined using a shake
flask method to show log D7.4 of 3.5 0.1, significantly higher
than that predicted by the in silico method (2.3), or
experimentally estimated using HPLC method (2.1).
chemical shift of 7.6 ppm upfield for sterically compressed
carbon atom Callylic in (E)-13. A similar chemical shift of 7.7
ppm units upfield was found for α-hydroxy ethyl oxime ether
(E)-20 for the Callylic carbon atom with a lower degree of steric
freedom (Figure 4a). Further confirmation of stereochemical
assignment was sought, and the NOESY experiments
performed for both geometrical isomers failed to detect NOE
between Callylic and the ethyl group from the oxime
functionality presumably due to significant distance between
the two. However, 1H NMR showed comparable shifts as those
seen in the fluorine case (Figure 2a). One possible explanation
for the preference of the E-isomer could be postulated by
invoking dipole−dipole interactions in 20 (Figure 4b). The E-
isomer configuration facilitates minimization of dipole−dipole
interactions.
At this juncture, we encountered another challenge of
separating the two isomers. While the E-isomer was easily
obtained in meaningful amounts, only trace amounts of Z-
isomer were isolated after the purification, and we therefore
selected the E-isomer of 16 for completion of the synthesis.
Ethyl oxime ether (E)-20 was coupled with 2-bromopyridine
under the Sonogashira reaction conditions to yield the alcohol
(E)-21, which was tosylated to afford (E)-22 in 38% yield
(Scheme 3). Both the Sonogashira cross-coupling and
tosylation were performed under basic conditions, but no
isomerization of the oxime ether double bond was observed.
This finding was further compounded when tosylate (E)-22
was heated in deuterated chloroform over 3 h at 60 °C to show
the full stability of the E-isomer.
The equilibrium dissociation constant Kd of (E)-[18F]-16 was
further explored in a Scatchard assay (see Supporting
Information). A Kd value of 70 nM was estimated for (E)-
[18F]-16 in a single experiment performed in triplicate which
was in complete agreement with the previously determined
IC50 (68 nM).
Radiolabeled (E)-[18F]-16 also served as a model compound
to establish if the binding was specific to mGluR5 in vitro. The
autoradiographic study was performed on rat brain slices using
1 or 10 nM solutions of (E)-[18F]-16 (Figure 5). Importantly,
the distribution of radioactivity was heterogeneous, and the
highest uptake was observed in regions where mGluR5 is highly
expressed in the brain (i.e., hippocampus and cortex).
Additionally, the brain slices were incubated with the
solution of (E)-[18F]-16 (1 or 10 nM) and cold 1 (100 nM).
A complete blockade of radioactivity was observed, indicating
that (E)-[18F]-16 specifically binds to mGluR5 in vitro (Figure
5).
CONCLUSIONS
■
In the search for a fluorine-18-labeled PET radiotracer for
imaging mGluR5, we have successfully synthesized and
structurally assigned five novel derivatives based on the
structural manifold of [11C]-1 by employing an enolization/
NFSI α-fluorination method. Our in vitro data enabled
identification of (Z)-16 as a potential mGluR5 PET radiotracer.
However, the synthesis of the tosylate precursor 22 revealed a
stereochemical preference for the E-isomer of α-hydroxy
derivative (E)-20 which prompted us to prepare (E)-[18F]-16
We next investigated the radiolabeling of the model tosylate
precursor (E)-22 in DMF using Kryptofix/18F− complex
(Scheme 4). The radiolabeling was successful, albeit in low
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obtained with a Waters Micromass AutoSpec Ultima MassLynx 4.0
spectrometer. Ion mass/charge (m/z) ratios are reported as values in
atomic mass units. Semipreparative purification of radiolabeled
material was performed on a Merck-Hitachi L6200A system equipped
with Knauer variable wavelength detector and an Eberline radiation
detector using a reverse phase column (C18 Phenomenex Gemini, 5
μm, 250 × 10 mm) and eluting with the following gradient: 0−5 min
5% aq MeCN, 5−15 min 5−50% aq MeCN, 15−30 min 50% aq
MeCN, 30−50 min, 50−90% aq MeCN, 50−65 min, 65% MeCN at
flow rate 5 mL/min. Analytical HPLC samples were analyzed by an
Agilent HPLC 1100 system equipped with a UV multiwavelength
detector and a Raytest Gabi star radiation detector using a reverse
phase column (ACE 111-0546, C18, 3 μm, 50 × 4.6 mm) and eluting
with 45% aq MeCN at flow rate 1 mL/min. Samples for PBS and
plasma stability were analyzed by HPLC using an Agilent 1100 system
with Gina software, equipped with UV multiwavelength and Raytest
Gabi Star detectors. For HPLC analysis, a reversed phase column
(Phenomenex, Gemini 10 μm C18 column, 300 × 3.9 mm,
Phenomenex) was used at 1 mL/min flow of 70% aq MeCN.
Trimethyl((5-((trimethylsilyl)ethynyl)cyclohexa-1,5-dien-1-
yl)oxy)silane (8). A one-neck round-bottom flame-dried flask was
charged at ambient temperature under N2 atmosphere with anhydrous
tetrahydrofuran (8 mL), diisopropylamine (0.77 mL, 554 mg, 5.49
mmol, d = 0.722) was added, and the colorless solution was cooled to
−20 °C (few pieces of dry ice in the acetone bath). The mixture was
then treated with n-butyllithium (3.5 mL, 4.99 mmol, c = 1.43 M
solution in hexanes) dropwise via syringe over 2 min, and the resulting
pale yellow solution was stirred for 36 min. After this time, the pale
yellow clear LDA solution was further cooled to −78 °C (dry ice/
acetone bath), and it was then treated with a solution of 3-
ethynylcyclohex-2-enone (500 mg, 4.16 mmol) in anhydrous
tetrahydrofuran (4 mL) dropwise via syringe over 9 min during at
which time the mixture turned brown (on the surface purple
coloration was observed) and a precipitate formed. The resulting
heterogeneous mixture was stirred at −78 °C under N2 atmosphere for
64 min. After this time, chlorotrimethylsilane (1.0 mL, 904 mg, 8.32
mmol, d = 0.856) was added in one portion, and the resulting mixture
was stirred at −78 °C under N2 for 64 min, during which time the
mixture turned bright orange and clear. After this time, the cooling
bath was removed, the orange clear mixture was allowed to warm to
ambient temperature over 30 min, and the crude mixture was then
poured over ice-cold 5% wt aq NaHCO3 (40 mL) and was diluted
with Et2O (50 mL). The two layers were shaken well and separated.
The organic phase was washed with H2O (3 × 35 mL) and brine (1 ×
35 mL), dried (Na2SO4), and concentrated in vacuo to give crude
material as a bright red oily residue (1.0 g, 3.79 mmol, 91%). The
crude mixture was used for the next step without further purification:
1H NMR (400 MHz, CDCl3) δ 6.06 (bd, J = 1.8 Hz, 1H), 5.02−4.96
(m, 1H), 2.24−2.20 (m, 4H), 0.20 (s, 9H), 0.19 (s, 9H) ppm.
6-Fluoro-3-((trimethylsilyl)ethynyl)cyclohex-2-enone (9). At
ambient temperature under N2 atmosphere, a one-neck round-bottom
flask was charged with the crude mixture of trimethyl((5-
((trimethylsilyl)ethynyl)cyclohexa-1,5-dien-1-yl)oxy)silane (1.08 g,
4.10 mmol), anhydrous dichloromethane (6 mL) was added, and
the resulting clear red mixture was cooled to −78 °C (dry ice/acetone
bath). The mixture was then treated with N-fluorodibenzenesulfoni-
mide (1.30 g, 4.10 mmol, 97% pure) portionwise (with each portion,
the flask was quickly opened to air) over 4 min, and the resulting
brown heterogeneous mixture was stirred under N2 and slowly
warmed to ambient temperature over 26.5 h. After this time, the crude
reaction mixture was quenched with 0.1 M aq HCl (7 mL), the
mixture was further diluted with H2O (7 mL) and CH2Cl2 (12 mL),
and the two layers were shaken well and separated. The aqueous phase
was extracted with CH2Cl2 (2 × 12 mL). The combined organic
extracts were dried (Na2SO4) and concentrated in vacuo to give crude
material as a red oily residue (2.56 g) which was purified by
chromatography on a silica gel column (eluting with 5% EtOAc/
pentane) to give the title compound (205 mg, 0.97 mmol, 24%) as a
pale yellow oil: IR (neat) 2962, 2904, 1696, 1591, 1430, 1354, 1253,
1203, 1154, 1096, 846, 765, 703 cm−1; 1H NMR (400 MHz, CDCl3) δ
Figure 5. Autoradiography on horizontal rat brain slices, indicating
heterogeneous distribution of activity with highest uptake in mGluR5-
rich regions. The first and second rows represent application of 1 and
10 nM [18F]-16, respectively, and the first and second column
represent baseline and blocking conditions, respectively.
as a model compound for the establishment of a radiolabeling
strategy and in vitro testing. We successfully radiosynthesized
(E)-[18F]-16 and demonstrated its stability in vitro in plasma
and PBS and specificity to mGluR5. Encouraged by the success
of fluorine-18 radiolabeling in an α-position of the oxime ether
double bond and the heterogeneous mGluR5 specific uptake of
(E)-[18F]-16 in rat brain slices in vitro, exploration of
alternative routes to access the Z-isomer selectively are
currently underway in our lab. The results of these studies
will be reported in due course.
MATERIALS AND METHODS
■
General Techniques. All reactions requiring anhydrous conditions
were conducted in flame-dried glass apparatus under an atmosphere of
inert gas. All chemicals and anhydrous solvents were purchased from
Aldrich or ABCR and used as received unless otherwise noted.
Reported density values are for ambient temperature. [3H]-1 (2.405
GBq/μmol, 37 MBq/mL solution in EtOH) was obtained from
AstraZeneca. Purity of compounds was ≥95% as determined by
analytical HPLC method on an Agilent HPLC system. Preparative
chromatographic separations were performed on Aldrich Science silica
gel 60 (35−75 μm) and reactions followed by TLC analysis using
Sigma-Aldrich silica gel 60 plates (2−25 μm) with fluorescent
indicator (254 nm) and visualized with UV or potassium
permanganate. Infrared spectra were recorded on a JASCO FT/IR
6200 (OmniLab) spectrometer using a chloroform solution of
compound. 1H and 13C NMR spectra were recorded in Fourier
transform mode at the field strength specified on Bruker Avance FT-
NMR spectrometers. Spectra were obtained from the specified
deuterated solvents in 5 mm diameter tubes. Chemical shift in ppm
is quoted relative to residual solvent signals calibrated as follows:
CDCl3 δH (CHCl3) = 7.26 ppm, δC = 77.2 ppm; (CD3)2SO δH
1
(CD3SOCHD2) = 2.50 ppm, δC = 39.5 ppm. Multiplicities in the H
NMR spectra are described as follows: s = singlet, d = doublet, t =
triplet, q = quartet, quint. = quintet, m = multiplet, b = broad; coupling
constants are reported in hertz. Numbers in parentheses following
carbon atom chemical shifts refer to the number of attached hydrogen
atoms as revealed by the DEPT spectral editing technique.
Electrospray (ES) mass spectra (LRMS) were obtained with a
Micromass Quattro micro API LC electrospray ionization, and
electrospray (ES) mass spectra (HRMS) were obtained with a Bruker
FTMS 4.7 T BioAPEXII spectrometer. Electron-impact (EI) and
chemical ionization (CI) mass spectra (LRMS and HRMS) were
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6.24−6.22 (m, 1H), 4.91 (ddd, J = 48.0, 12.2, 5.1 Hz, 1H), 2.67−2.60
(m, 2H), 2.47−2.16 (m, 2H), 0.23 (s, 9H) ppm; 13C NMR (100 MHz,
CDCl3) δ 193.3 (d, J = 15.3 Hz, 0), 143.4 (0), 131.4 (1), 108.2 (0),
102.6 (0), 89.7 (d, J = 186 Hz, 1), 29.4 (d, J = 12.6 Hz, 2), 29.3 (d, J =
3.6 Hz, 2), −0.31 (3) ppm; 19F NMR (376 MHz, CDCl3) δ −192.4
(dm, J = 49.3 Hz) ppm; MS (ES+) m/z 211 (M + H)+; HRMS (ESI)
m/z 211.0938 (calcd for C11H16FOSi: 211.0949).
mixture was stirred at ambient temperature for 22 h. After this time,
the brown mixture was quenched with saturated aq NH4Cl (8 mL)
and then diluted with H2O (3 mL) and EtOAc (11 mL), and the two
layers were shaken well and separated. The aqueous phase was
extracted with EtOAc (2 × 11 mL). The combined organic phase was
washed with H2O (3 × 8 mL) and brine (1 × 8 mL), dried (Na2SO4),
and concentrated in vacuo to give crude material as a brown oily
residue (338 mg) which was then purified by chromatography on a
silica gel column (eluting with gradient 20−30% EtOAc/pentane) to
give the title compound (20 mg, 0.08 mmol, 46%) as a pale yellow oil:
IR (neat) 2978, 2933, 1580, 1562, 1462, 1428, 1048, 987, 959, 891,
859, 779 cm−1; 1H NMR (400 MHz, CDCl3) δ 8.60 (ddd, J = 4.9, 1.7,
1.0 Hz, 1H), 7.66 (td, J = 7.7, 1.8 Hz, 1H), 7.45 (dt, J = 7.8, 1.1 Hz,
1H), 7.23 (ddd, J = 7.6, 4.9, 1.2 Hz, 1H), 6.59 (dm, J = 2.6 Hz, 1H),
5.86 (dt, J = 47.4, 2.5 Hz, 1H), 4.24 (ddm, J = 7.1, 5.8 Hz, 2H), 2.71−
2.59 (m, 1H), 2.43−2.26 (m, 2H), 1.79 (dm, J = 46.3 Hz, 1H), 1.31 (t,
J = 7.0 Hz, 3H) ppm; 13C NMR (100 MHz, CDCl3) δ 150.4 (d, J =
13.7 Hz, 0), 150.1 (1), 143.1 (0), 136.1 (1), 129.0 (d, J = 1.1 Hz, 1),
127.3 (1), 126.6 (d, J = 2.2 Hz, 0), 123.1 (d, J = 20.0 Hz, 1), 92.4 (0),
89.3 (0), 77.6 (d, J = 167 Hz, 1), 70.8 (3), 27.5 (d, J = 22.2 Hz, 1),
24.3 (d, J = 3.2 Hz, 1), 14.5 (3) ppm; 19F NMR (376 MHz, CDCl3) δ
− 188.4 (td, J = 47.0, 12.0 Hz) ppm; MS (ES+) m/z 259 (M + H)+;
HRMS (ESI) m/z 259.1240 (calcd for C15H16FN2O: 259.1241).
(E)-6-Fluoro-3-(pyridin-2-ylethynyl)cyclohex-2-enone O-
Ethyl Oxime ((E)-16). A method analogous to that described for
(Z)-16 was employed, starting with (E)-6-fluoro-3-((trimethylsilyl)-
ethynyl)cyclohex-2-enone O-ethyl oxime (48 mg, 0.19 mmol) to give
crude material as a brown oily residue which was then purified by
chromatography on a silica gel column (eluting with 20% EtOAc/
pentane) to give the title compound (18 mg, 0.07 mmol, 37%) as a
pale yellow oil: IR (neat) 2977, 2934, 1581, 1463, 1429, 1369, 1049,
6-Fluoro-3-((trimethylsilyl)ethynyl)cyclohex-2-enone O-
Ethyl Oxime (13). At ambient temperature under an Ar atmosphere,
a pear-shaped flask was charged with 6-fluoro-3-((trimethylsilyl)-
ethynyl)cyclohex-2-enone (150 mg, 0.71 mmol), pyridine (2.4 mL)
was added, and the resulting pale clear mixture was further treated with
O-ethylhydroxylamine hydrochloride (104 mg, 1.07 mmol) in one
portion. The resulting mixture was stirred at ambient temperature
under Ar for 21 h. After this time, the mixture was diluted with H2O (6
mL) and Et2O (10 mL), and the two layers were shaken well and
separated. The aqueous phase was extracted with Et2O (2 × 10 mL).
The combined organic extracts were washed with saturated aq CuSO4
(3 × 7 mL), H2O (1 × 10 mL), and brine (1 × 10 mL), dried
(Na2SO4), and concentrated in vacuo to give crude material as a pale
brown oil. The crude mixture was purified by chromatography on a
silica gel column (eluting with 2% EtOAc/pentane) to give (Z)-6-
fluoro-3-((trimethylsilyl)ethynyl)cyclohex-2-enone O-ethyl oxime
(86.7 mg, 0.34 mmol, 48%): IR (neat) 2958, 2146, 1360, 1250,
1051, 987, 843, 760 cm1; 1H NMR (400 MHz, CDCl3) δ 6.46 (dm, J
= 2.8 Hz, 1H), 5.83 (dt, J = 47.5, 2.8 Hz, 1H), 4.22 (ddm, J = 7.1, 6.0
Hz, 2H), 2.61−2.59 (m, 1H), 2.33−2.20 (m, 2H), 1.73 (dm, J = 46.4
Hz, 1H), 1.30 (t, J = 7.1 Hz, 3H), 0.21 (s, 9H) ppm; 13C NMR (100
MHz, CDCl3) δ 150.7 (d, J = 13.7 Hz, 0), 128.5 (d, J = 1.2 Hz, 1),
127.6 (d, J = 2.3 Hz, 0), 105.4 (0), 99.1 (0), 77.8 (d, J = 167 Hz, 1),
70.8 (2), 27.7 (d, J = 22.3 Hz, 2), 24.6 (d, J = 3.2 Hz, 2), 14.7 (3), 0.0
(3) ppm; 19F NMR (376 MHz, CDCl3) δ − 188.5 (td, J = 47.0, 12.4
Hz) ppm; MS (ES+) m/z 254 (M + H)+; HRMS (ESI) m/z 254.1373
(calcd for C13H21FNOSi: 254.1371) and (E)-6-fluoro-3-
((trimethylsilyl)ethynyl)cyclohex-2-enone O-ethyl oxime (46.8 mg,
0.18 mmol, 26%): IR (neat) 2960, 2142, 1595, 1573, 1428, 1250,
1
977, 912, 890, 781, 742 cm−1; H NMR (400 MHz, CDCl3) δ 8.62
(ddd, J = 4.8, 1.7, 0.9 Hz, 1H), 7.68 (td, J = 7.7, 1.8 Hz, 1H), 7.47 (dt,
J = 7.8, 1.1 Hz, 1H), 7.25 (ddd, J = 7.6, 4.9, 1.2 Hz, 1H), 7.14 (dm, J =
2.7 Hz, 1H), 5.15 (dm, J = 50.1 Hz, 1H), 4.23 (qd, J = 7.1, 0.6 Hz,
2H), 2.77−2.64 (m, 1H), 2.51−2.33 (m, 2H), 1.91 (dm, J = 43.3 Hz,
1H), 1.31 (t, J = 7.1 Hz, 3H) ppm; 13C NMR (100 MHz, CDCl3) δ
150.4 (1), 148.3 (d, J = 16.6 Hz, 0), 143.0 (0), 136.4 (1), 129.8 (0),
127.6 (1), 123.4 (1), 121.8 (1), 93.6 (0), 89.3 (0), 86.5 (d, J = 170 Hz,
1), 70.7 (2), 28.4 (d, J = 22.6 Hz, 2), 25.5 (d, J = 4.7 Hz, 2), 14.6 (3)
ppm; 19F NMR (376 MHz, CDCl3) δ − 178.4 (ddd, J = 50.7, 43.2, 8.3
Hz) ppm; MS (ES+) m/z 259 (M + H)+; HRMS (ESI) m/z 259.1236
(calcd for C15H16FN2O: 259.1241).
1
1049, 975, 842, 760, 638 cm−1; H NMR (400 MHz, CDCl3) δ 6.96
(dm, J = 2.7 Hz, 1H), 5.11 (dm, J = 49.2 Hz, 1H), 4.21 (q, 7.0 Hz,
2H), 2.66−2.53 (m, 1H), 2.39−2.28 (m, 2H), 1.85 (dm, J = 43.2 Hz,
1H), 1.30 (t, J = 7.1 Hz, 3H), 0.22 (s, 9H) ppm; 13C NMR (100 MHz,
CDCl3) δ 148.5 (d, J = 16.7 Hz, 0), 130.6 (d, J = 2.8 Hz, 0), 120.9 (1),
105.2 (0), 100.7 (0), 86.6 (d, J = 169 Hz, 1), 70.6 (2), 28.3 (d, J = 22.8
Hz, 2), 25.8 (d, J = 4.8 Hz, 2), 14.7 (3), 0.0 (3) ppm; 19F NMR (376
MHz, CDCl3) δ − 178.5 (ddd, J = 50.4, 43.2, 7.9 Hz) ppm; MS (ES+)
m/z 254 (M + H)+; HRMS (ESI) m/z 254.1375 (calcd for
C13H21FNOSi: 254.1371).
X-ray Analysis: Crystal Data for (Z)-17. C16H17FN2O, MW
=
272.32, orthorhombic space group P212121, a = 6.2820(1) Å, b =
11.4820(3) Å, c = 19/6867(5) Å; data measurement performed on a
Bruker APEX II Duo diffractometer at 100 K, radiation Mo Kα (λ =
0.71073 Å), z = 4, R = 0.032 for 3090 I > 2σ(I), 0.033 for all 3248
unique reflections, GOF = 1.16. Full crystallographic data have been
deposited with the Cambridge Crystallographic Data Centre under
deposition number CCDC 880418 and can be obtained free of charge
via www.ccdc.cam.ac.uk/.
(Z)-6-Fluoro-3-(pyridin-2-ylethynyl)cyclohex-2-enone O-
Ethyl Oxime ((Z)-16). At ambient temperature under a N2
atmosphere, a one-neck pear-shaped flask was charged with (Z)-6-
fluoro-3-((trimethylsilyl)ethynyl)cyclohex-2-enone O-ethyl oxime (43
̀
mg, 0.17 mmol), anhydrous N,N-dimethylformamide (0.8 mL) was
added, and the resulting pale yellow solution was further treated with
tetrabutylammonium fluoride solution in tetrahydrofuran (0.34 mL,
0.34 mmol, c = 1 M) dropwise over 1 min during which time the
mixture turned brown. Then the mixture was stirred for 13 min. TLC
analysis indicated that all starting material was consumed, and without
workup and/or further purification, the mixture was used for the next
step. A two-neck round-bottom flask was evacuated at ambient
temperature, and it was then backfilled with N2, repeating this three
times. This flask was then charged with the crude reaction mixture of
(Z)-3-ethynyl-6-fluorocyclohex-2-enone O-ethyl oxime (31 mg, 0.17
mmol, still containing TBAF·THF and DMF), and additional
(E)-6-Fluoro-3-(pyridin-2-ylethynyl)cyclohex-2-enone O-
Ethyl Oxime ((E)-[18F]-16). No-carrier-added [18F]-fluoride was
produced via nuclear 18O(p, n)18F reaction from enriched 18O-water
using an IBA cyclone 18/9 cyclotron, and it was immediately trapped
on a QMA cartridge (preconditioned with 0.5 M aq K2CO3 (1 × 5
mL) and then H2O (1 × 5 mL) and dried in air). The trapped [18F]-
fluoride was eluted from the cartridge with 0.25 wt % Kryptofix-222
solution (1 mL) in basic (0.05 wt % K2CO3) aq MeCN (75% vv) into
a tightly closed reaction vial. The solvents were evaporated in vacuo
(130 mbar) with a gentle stream of N2 gas at 110 °C over 5 min. To
the resulting solid residue was then added anhydrous MeCN (1 mL),
and the mixture was azeotropically dried in vacuo (130 mbar) with a
gentle stream of N2 at 110 °C. To the dried Kryptofix-222/[18F−]
complex was added a solution of (E)-22 (2.19 mg, 5.33 μmol) in
̀
anhydrous N,N-dimethylformamide (1.5 mL) was used for quantita-
tive transfer. The resulting red mixture was further treated with 2-
bromopyridine (16 μL, 27.0 mg, 0.17 mmol, d = 1.657) followed by
triethylamine (0.28 mL, 206 mg, 2.04 mmol, d = 0.726), and then
copper(I) iodide (3.2 mg, 17.0 μmol) was added in one portion (flask
was quickly opened to the air). Finally tetrakis(triphenylphosphine)-
palladium(0) (6.0 mg, 5.1 μmol) was added in one portion (the flask
was once again opened to air quickly), and the resulting brown
̀
anhydrous N,N-dimethylformamide (0.3 mL), and the dark brown
mixture was heated at 90 °C for 13 min. The crude mixture was
diluted with 50% v/v aq MeCN (2 mL) and purified via
semipreparative HPLC. The desired product was collected (retention
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Journal of Medicinal Chemistry
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time: 30.9 min) and immediately diluted with H2O (10 mL). The
aqueous solution was passed through a C18 cartridge (preconditioned
with EtOH (1 × 5 mL) and then H2O (1 × 5 mL) and dried in air),
the cartridge was washed with H2O (2 × 1 mL), and the product was
eluted from the C18 cartridge with EtOH (1 × 0.3 mL) into a sterile
vial containing 50% aq PEG200 (5 mL) to afford the radiolabeled title
compound in a 3% decay-corrected yield. Typically, starting from ca.
35 GBq of activity, 740 MBq of product was obtained. The
radiochemical purity was >99%, and specific activity was 30 GBq/
μmol.
Competition Binding Assay. Brain membranes were prepared
from Sprague−Dawley rat brains as described previously.40 Frozen
membranes were thawed on ice and pelleted at 45000g at 4 °C 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 °C) and resuspended in HEPES buffer at a protein
concentration of 1.3 mg/mL. The binding assay was performed as
previously described.40 In brief, brain membranes (0.1 mg of protein)
were incubated in triplicate at ambient temperature with 2 nM [3H]-1
and (E)-16 at concentrations between 10 pM and 100 μM in a total
volume of 0.2 mL of HEPES. (E)-16 was diluted from a 1 mM
ethanolic (50%) solution. The corresponding EtOH concentrations
did not affect [3H]-1 binding (data not shown). Nonpecific binding of
[3H]-1 was estimated with 100 μM MMPEP. After 45 min, the
samples were filtered, and the filters containing the membranes with
bound [3H]-1 were measured in a β-counter (Beckman LS6500).
Bound [3H]-1 (B, pmol per mg protein) was fitted with Excel solver to
eq 1 to estimate IC50.
Author Contributions
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors acknowledge Mrs. Petra Wirth and Dr. Adrienne
Muller for technical support. Professor Paul R. Blakemore
̈
(Oregon State University) is acknowledged for helpful
discussions on NMR assignments. Drs. Bernhard Pfeiffer
(Altman group) and Marc-Olivier Ebert (Laboratory for
Organic Chemistry, ETH) are acknowledged for technical
support with NMR analysis. Dr. Ebert is acknowledged for
performing additional NOE experiments for compounds (E)-
20 and (Z)-20. Ms. Sara Wyss (Werner group) is acknowl-
edged for performing chiral GC analysis of ent-9. Dr. Mark A.
Sephton (ZHAW) is acknowledged for proofreading the
manuscript. Ms. Cindy Fisher, Drs. Cindy A. Wanger-Baumann,
and Thomas Betzel are acknowledged for useful discussions.
ABBREVIATIONS USED
■
mGluR5, metabotropic glutamate receptor subtype 5; PET,
positron emission tomography; NFSI, N-fluorodibenzenesulfo-
nimide; mCPBA, m-chloroperbenzoic acid; DMF, N,N′-
dimethylformamide; THF, tetrahydrofuran; [11C]-ABP688,
(E)-3-((6-methylpyridin-2-yl)ethynyl)cyclohex-2-enone O-
[11C]methyl oxime; [3T]-ABP688, (E)-3-((6-methylpyridin-2-
yl)ethynyl)cyclohex-2-enone O-[3T]methyl oxime; [18F]-
SP203, 3-fluoro-5-(2-([18F](fluoromethyl)thiazol-4-yl)-
ethynyl)benzonitrile; [18F]-FPEB, 3-[18F]fluoro-5-(2-
pyridinylethynyl)benzonitrile; [18F]-FDEGPECO, (E)-3-(pyr-
idin-2-ylethynyl)cyclohex-2-enone O-(2-(2-[18F]¯uoroethoxy)-
ethyl) oxime; MMPEP, 2-[(3-methoxyphenyl)ethynyl]-6-meth-
ylpyridine; PBS, phosphate buffer in saline; SAR, structure−
activity relationship; TLC, thin layer chromatography; HPLC,
high pressure liquid chromatography; GC, gas chromatography;
IC50, half maximal inhibitory concentration; Ki, inhibition
constant; Kd, dissociation constant
B = Bmin + ((Bmax − Bmin)/(1 + (C/IC50)))
(1)
where C is the total (E)-16 concentration, Bmax is the maximal B, i.e.,
the plateau in the B/C plot at low log C and Bmin is the minimal B, i.e.,
the plateau at high log C. The inhibition constant Ki of (E)-16 was
estimated from the IC50 and Kd of [11C]-1 (1.7 0.2 nM)9 with the
Cheng−Prusoff equation.
Stability in PBS and Plasma. (E)-[18F]-16 (21 MBq) was
incubated in phosphate buffer (4 mM KH2PO4/Na2HPO4, 155 mM
NaCl, pH 7.4) or rat plasma at 37 °C for up to 2 h. At different time
points, samples were61−63 diluted and reactions stopped with ice-cold
MeCN (140 μL). Plasma samples were centrifuged at 12000g for 10
min. The samples were filtered, and supernatants were analyzed by
HPLC.
In Vitro Autoradiography. Frozen horizontal brain slices (20
μm) from a male Wistar rat (221 g) adsorbed to SuperFrost Plus slides
were thawed at ambient temperature and preincubated 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 or 10 nM (E)-[18F]-16 alone or together with 100
nM 1 in HEPES buffer for 45 min at ambient temperature. After
incubation, the solutions were decanted and the slides washed on ice
in HEPES buffer containing 0.1% BSA and twice in HEPES buffer (3
min each) and finally dipped in H2O. Dried slides were exposed to a
phosphor imager plate for 30 min, and the plate was scanned in a
BAS5000 reader (Fuji).
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Full experimental procedures and characterization of all
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NOTE ADDED AFTER ASAP PUBLICATION
■
This manuscript was published ASAP on August 9, 2012. Due
to a production error, additional corrections were made to
Table 1 and the revised version was reposted on August 14,
2012.
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7162
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