DISS. ETH 18915
Development of Novel Fluorine-18 Labeled PET Tracers for Imaging of
the Metabotropic Glutamate Receptor Subtype 5 (mGluR5)
A dissertation submitted to
ETH ZURICH
for the degree of
DOCTOR OF SCIENCES
presented by
CINDY ANNA BAUMANN
Pharmacist
University of Regensburg
Born 24.08.1981
German citizen
accepted on the recommendation of
Prof. Dr. P. August Schubiger , examiner
Prof. Dr. Simon M. Ametamey, co-examiner
Prof. Dr. Hanns U. Zeilhofer, co-examiner
2010
Herzlichen Dank!
Herzlicher Dank gebührt allen, die mich in den letzten drei Jahren in verschiedenster
Weise so tatkräftig unterstützt haben und zum Gelingen dieser Doktorarbeit beigetragen
haben. Dabei danke ich ganz besonders Herrn Professor Schubiger für die grosse Chance,
in seiner Gruppe an der ETH promovieren zu koennen. Zusammen mit Simon möchte ich
ihm für das grossartige Projekt danken, das mir nahezu an jedem einzelnen Tag Freude
bereitet hat. Ganz besonders Simon habe ich es zu verdanken, dass ich mich für das ZNZ
Programm begeisterte. Es hat mir mitunter die schönsten Erfahrungen während der
Dissertation gebracht und führte mich zu Professor Zeilhofer, der zum Dritten im Bunde
des Thesis Steering Commitees wurde. Dafür danke ich Uli!
Ganz zu Beginn waren es vor allem Christophe und Claudia, die mir die wichtigsten
ersten Schritte im Labor gezeigt haben, von denen ich über die ganze Zeit profitierte, und
wofür ich ihnen dankbar bin. Von Anfang an war Michael für die Vermittlung der
pharmakologischen Aspekte des Projektes an mich zuständig, und ich danke ihm für die
Weitergabe seines Wissens. Tom hat mir immer Mut gemacht und viel mit mir über
Chemie diskutiert. Ich danke ihm für seine Unterstützung. Stefanie war ebenfalls eine
solche Motivationsquelle, weil sie selbst immer ein Beispiel an Tatkraft war. Ich bedanke
mich bei ihr für die interessanten Diskussionen und ihren Rat. Linjing bin ich dankbar,
weil sie mir die Radiosynthesen beigebracht hat. Zu einer Art „Doktormutter“ machte sie
aber vor allem ihr guter Rat in jeder Lebenslage und ihr Vertrauen in mich. Das war für
mich etwas Besonderes. Von Matze konnte ich ebenfalls Vieles lernen. Ich schulde ihm
grossen Dank, für seine technische Unterstützung, seine Tatkraft und seine Freundschaft,
die er mir unabhängig von äusseren Umständen zukommen liess. Wir hatten zusammen
mit Norbert, der leider wieder nach Wien musste, viele lustige Abende.
Den vier Projektarbeits- und Masterstudenten, Sinja, Lukas, Nicole und Patrick, die ich
mit Freude betreuen durfte, danke ich recht herzlich. Ausserdem bin ich Benny und
Evgeny dankbar. Ich bedanke mich bei Matthias. Den Doktoranden und allen
Mitarbeitern der Schubiger Gruppe sowie Sara Belli möchte ich danken für ihre
Zusammenarbeit und ihre Kollegialität. Besonders Marian, Cindy und Petra haben viel
beigetragen. Weiterhin bin ich Monika dankbar für das Managment meiner gesamten ITProbleme. Harriet mit ihrer enormen Hilfsbereitschaft während des Schreibens gebührt
ein dickes Dankeschön.
Contents
Summary
9
Zusammenfassung
13
Abbreviations
17
1. Introduction
1.1 Positron Emission Tomography…………………………………………………23
1.2 Glutamate Receptors…………………………………………………………….30
1.3 Aim of the Thesis………………………………………………………………..41
1.4 References……………………………………………………………………….42
2. “Structure-Activity Relationships of Fluorinated ABP688 Derivatives
and the Discovery of a High Affinity Analogue as a Potential
Candidate for Imaging MGluR5 with PET”
2.1 Abstract………………………………………………………………………….53
2.2 Introduction……………………………………………………………………...53
2.3 Chemistry………………………………………………………………………..56
2.4 Results and Discussion…………………………………………………………..62
2.5 Conclusion……………………………………………………………………….66
2.6 Experimental Section……………………………………………………………67
2.7 References……………………………………………………………………….78
3. “Syntheses and Pharmacological Characterization of Novel Thiazole
Derivatives as Potential MGluR5 PET Ligands“
3.1.Abstract………………………………………………………………………….87
3.2.Introduction……………………………………………………………………...87
3.3.Results…………………………………………………………………………...90
3.4.Discussion……………………………………………………………………….98
3.5.Conclusion……………………………………………………………………..100
3.6.Experimental…………………………………………………………………...101
3.7.References……………………………………………………………………...111
4. “In vitro and in vivo Evaluation of [18F]-FDEGPECO as a PET
Tracer for Imaging the Metabotropic Glutamate Receptor Subtype 5
(MGluR5)”
4.1.Abstract………………………………………………………………………...119
4.2.Introduction………………………………………………………………….....120
4.3.Materials and Methods…………………………………………………………121
4.4.Results………………………………………………………………………….125
4.5.Discussion……………………………………………………………………...131
4.6.References……………………………………………………………………...133
5. Conclusions and Future Perspectives………………………………..137
Curriculum Vitae
141
Publications
143
Oral Presentations
144
Poster Presentations
145
Awards
146
9
Summary
Intensive investigations during the last years have shown that metabotropic glutamate
receptors are involved in numerous neurodegenerative diseases such as M. Parkinson, M.
Alzheimer and M. Huntington as well as other central nervous system disorders including
neurogenic pain, epilepsy, depression and drug addiction. The neurobiological basis of
these impairments is, however, still not well understood.
Non-invasive imaging techniques such as positron emission tomography (PET) offer the
possibility to visualize and analyze CNS receptors such as mGluR5 under various
physiological and pathophysiological conditions. Although the PET technology is well
advanced, the imaging of mGluR5 in humans is limited by the lack of high-affinity and
selective PET radioligands. Very recently, our group reported on the development of the
carbon-11 labeled PET ligand, [11C]-ABP688 with which the first imaging of mGluR5 in
humans succeeded. Carbon-11 has a relatively short physical half-life of 20 min.
Fluorine-18, however, has the best imaging characteristics of all PET radionuclides due to
its low positron energy and in addition, its half-life of 110 min allows for complex
synthesis and longer in vivo investigations. Most importantly, the long physical half-life
of fluorine-18 enables “satellite” distribution to clinical PET centers not equipped with
radiochemistry facilities.
Prompted by the need to develop a fluorine-18 labeled PET radiotracer for imaging
mGluR5 in vivo, a series of fluorinated compounds based on the core structure of
ABP688 were synthesized. The candidates were designed such that the fluorine atom is
conveniently placed to allow a one-step radiosynthesis with fluorine-18. ABP688 consists
of a linker that connects a pyridine moiety with a cyclohexenoneoxime moiety.
Accordingly, two general concepts for the introduction of fluorine were considered. In the
first approach, possibilities for the introduction of fluorine at the aromatic moiety of the
molecules represented in ABP688 by a pyridine ring were considered. Therefore ABP688
analogues in which the position of the nitrogen atom in the pyridine ring was shifted and
several non-fluorinated and fluorinated derivatives in which the pyridine ring was
replaced by a thiazole moiety were synthesized. The second approach entailed the
replacement of the methyl group at the oxime functionality in ABP688 by ethoxy fluorine
containing aliphatic side chains, fluoropyridines and fluorobenzonitriles.
10 In total, 17 novel candidates were synthesized by convergent syntheses involving
Sonogashira coupling. The compounds were obtained in overall good to optimal yields
and were fully characterized by mass spectroscopy and NMR spectroscopy. Each
molecule was evaluated for its binding affinity towards mGluR5 in competition binding
assays. The binding studies resulted in six promising candidates having Ki values below
10 nM. Two compounds (E)-3-((2-(fluoromethyl)thiazol-4-yl)ethynyl)cyclohex-2-enone
O-methyloxime (FTECMO) and (E)-3-(pyridin-2-ylethynyl)cyclohex-2-enone O-2-(2fluoroethoxy)ethyl oxime (FDEGPECO) with Ki values of 5.5 ± 1.1 nM and 3.8 ± 0.4
nM, respectively, emerged as the most promising candidates. Both compounds were
therefore chosen and their utility as potential imaging agents for the mGluR5 was
investigated.
Due to easier synthetic accessibility FTECMO was initially investigated. The
radiosynthesis of [18F]-FTECMO was accomplished via nucleophilic substitution on a
bromo precursor in acetonitrile at 90 °C in 10 min to afford the final product in 45%
radiochemical yield and a specific activity of up to 120 GBq/µmol. [18F]-FTECMO
displayed optimal lipophilicity (logDpH7.4 = 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 heterogeneous and displaceable uptake of radioactivity in
mGluR5-rich brain regions such as striatum, hippocampus and cortex, while the
cerebellum a region with negligible mGluR5 expression exhibited low uptake of
radioactivity. In vivo PET studies were carried out subsequently in order to investigate the
in vivo distribution pattern of [18F]-FTECMO. PET imaging with [18F]-FTECMO in a
Wistar rat, however, revealed a low brain uptake. Accumulation of radioactivity in the
skull was also observed suggesting in vivo radiodefluorination. 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.
Attention was then shifted to FDEGPECO, the second promising candidate with high
binding affinity to mGluR5. FDEGPECO was radiolabeled in a single step with fluorine18 via nucleophilic substitution on the corresponding tosyl precursor in acetonitrile at
90 °C. The in vitro characterization of [18F]-FDEGPECO included stability studies in
human and in rodent plasma as well as in human liver microsomes. Scatchard analysis
confirmed the high binding affinity (KD1 = 0.61 ± 0.19 nM and KD2 = 13.73 ± 4.69 nM)
of [18F]-FDEGPECO. The novel radioligand was shown not to be a substrate for P-
11
glycoprotein (P-gp). In vitro autoradiographical studies with rat brain slices showed a
heterogeneous and displaceable uptake of radioactivity in mGluR5-rich brain regions
such as hippocampus and striatum. Subsequent PET imaging in Wistar rats revealed the
same radioactivity uptake pattern in the brain with highest accumulation in the
hippocampus and lowest in the cerebellum. In vivo blockade PET studies with M-MPEP
(1 mg/kg) resulted in reduced and homogeneous uptake of radioactivity in the rat brain,
indicating the specificity of [18F]-FDEGPECO for mGluR5. Post mortem biodistribution
studies also confirmed the distribution pattern observed in the PET images obtained with
[18F]-FDEGPECO in the rat brain. In the control group, mGluR5-rich brain regions such
as hippocampus, striatum and cortex showed highest radioactivity uptake while low levels
of radioactivity accumulation were obtained in the cerebellum and the brain stem. The
hippocampus-to-cerebellum and the striatum-to-cerebellum ratios were 2.57 and 2.89,
respectively. In the peripheral organs, highest radioactivity uptake was detected in the
liver indicating that the compound is cleared mainly via the hepatobiliary pathway.
Analysis of selected brain regions of the animals after co-injection with [18F]FDEGPECO and M-MPEP (1mg/kg) revealed a 50% specifictiy of the new ligand in
mGluR5-rich brain regions again confirming the in vivo specificity of [18F]-FDEGPECO
for mGluR5.
In conclusion, this project led to the discovery of six mGluR5 ligands with binding
affinities lower than 10 nM. Two of these candidates were successfully investigated for
their suitability as in vivo mGluR5 PET tracers. [18F]-FTECMO exhibited promising in
vitro properties but was plagued by in vivo defluorination. The second candidate, [18F]FDEGPECO, showed not only excellent in vitro properties but also good in vivo
properties. This study demonstrated that [18F]-FDEGPECO can be used as a PET tracer
for imaging mGluR5 in the rodent brain and may also be useful for imaging mGluR5 in
species such as monkeys and human subjects.
12 13
Zusammenfassung
In den letzten Jahren haben intensive Untersuchungen gezeigt, dass der metabotrope
Glutamatrezeptor vom Subtyp 5 (mGluR5) an der Entstehung von zahlreichen
neurodegenerativen Erkrankungen wie Morbus Parkinson, Morbus Alzheimer und
Morbus Huntington, so wie bei anderen Störungen des zentralen Nervensystems wie
neurogenem Schmerz, Epilepsie, Depressionen oder Drogenabhängigkeit beteiligt ist.
Allerdings sind die zugrunde liegenden neurobiologischen Abläufe und die genaue Rolle
von mGluR5 bezüglich dieser Störungen weitgehend ungeklärt.
Nicht invasive bildgebende Verfahren wie die Postitronen Emissions Tomographie (PET)
ermöglichen die Darstellung und die Analyse von ZNS-Rezeptoren und ebenso von
mGluR5 unter verschiedenen physiologischen und phatophysiologischen Bedingungen.
Allerdings bleibt die Darstellung von mGluR5 im Menschen wegen fehlender hoch
affiner und selektiver PET Radioliganden stark eingeschränkt, obwohl die PET
Technologie an sich schon weit entwickelt ist. Kürzlich veröffentlichte unsere
Arbeitsgruppe den mit Kohlenstoff-11 markierten PET Liganden [11C]-ABP688 und die
ersten erfolgreichen PET Bilder von mGluR5 im Menschen, die mit diesem Tracer
aufgenommen wurden. Allerdings hat Kohlenstoff-11 eine relativ kurze Halbwertszeit
von nur etwa 20 Minuten. Dahingegen hat das Fluor-18 wegen seiner tiefen
Positronenenergie von 0.64 MeV (im Vergleich zur Positronenenergie von Kohlenstoff11 von 0.96 MeV) die besten bildgebenden Eigenschaften unter allen PET
Radionukliden. Zusätzlich ermöglicht seine Halbwertszeit von 110 Minuten komplexe
Radiosynthesen sowie zeitaufwändige in vivo Untersuchungen. Weitherhin spricht für das
Nuklid Fluor-18 die Tatsache, dass die lange physikalische Halbwertszeit die
systematische Verteilung von
18
F-markierten PET-Tracern an klinische Zentren
ermöglicht, die über keine geeigneten radiochemischen Einrichtungen verfügen.
Die dadurch bedingte hohen Nachfrage nach einem Flour-18 markierten PET Radiotracer
für mGluR5, führte zur Synthese verschiedene fluorierter Verbindungen, die auf der
Grundstruktur von ABP688 basieren. Die Moleküle wurden so konzipiert, dass das
Fluoratom an einer Stelle im Molekül platziert ist, an der die Radiomarkierung in nur
einem Syntheseschritt möglich ist. ABP688 besteht aus einem Linker, der eine PyridinTeilstruktur mit einem Cyclohexenon-Oxim verbindet. Dementsprechend wurden zwei
generelle Konzepte für die Einführung von Fluor in das Molekül erdacht. Zunächst sollte
14 Fluor an der Pyridin-Teilstruktur eingefügt werden. Dafür wurden ABP688 analoge
Verbindungen synthetisiert, in denen die Position des Stickstoffatoms im Pyridinring
verändert war. Wegen der Abnahme der Bindungsaffinität dieser Leitstrukturen, wurden
fluorierte und nicht fluorierte Derivate hergestellt, in denen der Pyridinring durch
verschiedene Thiazole ersetzt wurde. Zur Verwirklichung des zweiten Konzepts wurde
die Methylgruppe an der Oxim-Teilstruktur von ABP688 durch verschiedene
Fluorobenzonitrile, Fluoropyridine oder Seitenketten, die eine Fluoroethoxy-Teilstruktur
aufwiesen, ersetzt.
Insgesamt wurden 17 neue Substanzen auf dem Weg der konvergenten Synthese erhalten,
die hauptsächlich unter Anwendung von Sonogashira Kreuzkopplungsreaktionen
synthetisiert wurden. Die Endverbindungen konnten in guter bis optimaler Ausbeute
hergestellt werden. Die Charakterisierung erfolgte mit Hilfe von Massenspektrometrie
und NMR-Spektroskopie. Zur Ermittlung der Bindungsaffinitäten zum mGluR5 wurde
jedes Molekül Kompetitionsexperimenten unterzogen. Die Bindungsstudien führten zu
sechs vielversprechenden Kandidaten, die Ki Werte unter 10 nM erreichten. Die
Verbindungen
methyloxim
(E)-3-((2-(fluoromethyl)thiazol-4-yl)ethynyl)cyclohex-2-enon
(FTECMO)
und
(E)-3-(pyridin-2-ylethynyl)cyclohex-2-enon
OO-2-(2-
fluoroethoxy)ethyl oxim (FDEGPECO) mit den Ki Werten von 5.5 ± 1.1 nM
beziehungsweise 3.8 ± 0.4 nM waren die interessantesten Kandidaten. Daher wurden
beide Verbindungen für weitere Untersuchungen ausgewählt, in denen ihre Eignung als
mGluR5 PET-Tracer geprüft wurde.
Aufgrund der einfacheren Herstellung von FTECMO und [18F]-FTECMO wurde dieser
Tracer zuerst untersucht. Die Radiosynthese von [18F]-FTECMO erfolgte durch
nukleophile Substitution an einem dafür konzipierten Bromprecursor in Acetonitril bei
90°C während 10 Minuten, was mit einer radiochemischen Ausbeute von 45% zum
gewünschten Endprodukt mit einer spezifischen Aktivität von bis zu 120 GBq/µmol
führte. In entsprechenden Experimenten wurde gezeigt, dass [18F]-FTECMO optimale
Lipophilie (logDpH7.4 = 1.6 ± 0.2) besitzt und in Plasma von Ratten, in humanem Plasma
sowie in Rattenlebermikrosomen ausreichend stabil ist. In vitro Autoradiographien mit
[18F]-FTECMO
liessen
eine
heterogene
und
verdrängbare
Anreicherung
von
Radioaktivität in den Hirnregionen mit hoher mGluR5 Expression wie dem Striatum, dem
Hippocampus und dem Cortex erkennen. Im Cerebellum, einer Hirnregion mit
vernachlässigbarer mGluR5 Konzentration, fand sich hingegen nur eine kleine Menge
15
von Radioaktivität. Weiterhin sollten PET Studien durchgeführt werden, um das
spezifische Verteilungsmuster von[18F]-FTECMO in vivo zu untersuchen. Ein erstes PET
Experiment in einer Wistar Ratte zeigte jedoch nur geringe Aufnahme von Radioaktivitiät
in das Hirn des Tieres. Dafür wurde jedoch eine Anreicherung von Radioaktivität in den
Knochen und im Schädel beobachtet, was auf Radiodefluorierung in vivo hinweisst.
Somit sind die in vivo Eigenschaften von [18F]-FTECMO trotz der ausgezeichneten
Eignung zur Detektion von mGluR5 in vitro unzureichend für die mGluR5 Bildgebung in
Ratten.
Daher konzentrierte man sich auf die zweite vielversprechende Verbindung, die noch
höhere Bindungsaffinität zu mGluR5 aufwiess: FDEGPECO. Die Markierung mit Fluor18 wurde ebenfalls in einem Schritt via nukleophiler Substitution an einem
entsprechenden Tosylprecursor in Acetonitril bei 90°C erreicht. Die in vitro
Charakterisierung von [18F]-FDEGPECO beinhaltete Stabilitätsstudien in Rattenplasma
und in humanem Plasma so wie in humanen Lebermikrosomen. Scatchard Experimente
bestätigten die hohe Bindungsaffinität (KD1 = 0.61 ± 0.19 nM und KD2 = 13.73 ± 4.69
nM) von [18F]-FDEGPECO und mit weiteren in vitro Versuchen wurde eindeutig gezeigt,
dass der neue Radioligand kein Substrat für das P-Glykoprotein (P-gp) ist. In vitro
autoradiographische Untersuchungen an Rattenhirnschnitten zeigten eine heterogene und
verdrängbare Aufnahme von Radioaktivitiät in den mGluR5-reichen Hirnregionen wie
Hippocampus, Striatum und Cortex. Die nachfolgenden PET Experimente in Wistar
Ratten wiessen das gleiche Muster von aufgenommener Radioaktivität auf, bei dem die
höchste Anreicherung im Hippocampus und die geringste im Cerebellum detektiert
wurde. Blockade PET Studien mit M-MPEP (1 mg/kg) resultierten in einer reduzierten
und homogenen Aufnahme von Radioaktivität in das Hirn der Ratten, was ein Indiz für
die
Spezifität
von
[18F]-FDEGPECO
für
mGluR5
ist.
Post
mortem
Biodistributionsstudien bestätigten das bisher beobachtete Verteilungsmuster, dass nach
Applikation von [18F]-FDEGPECO im Rattenhirn entsteht. Das Verhältnis der
angereicherten Radioaktivität im Hippocampus bzw. im Striatum im Vergleich zur
angereicherten Radioaktvitität im Cerebellum war 2.57 und 2.89. In den peripheren
Organen wurde die höchste Aufnahme von Radioaktivität in der Leber gemessen, was auf
eine hepatobiliäre Clearance des Radioliganden hindeutet. Die Analyse von ausgewählten
Hirnregionen derjeniger Tiere, die [18F]-FDEGPECO und M-MPEP (1 mg/kg)
gemeinsam verabreicht bekommen hatten, zeigte auf, dass eine etwa 50 %ige Spezifität
16 des neuen Radioliganden vorlag, was nochmals die Beobachtungen bezüglich der in vivo
Selektivität von [18F]-FDEGPECO für mGluR5 bestätigte.
Abschliessend ist zu honorieren, dass dieses Projekt zur Entdeckung von insgesamt sechs
mGluR5-Liganden geführt hat, die Bindunsaffinitäten mit Ki-Werten unter 10 nM
aufweisen. Zwei dieser Kandidaten wurden auf ihre Eignung als in vivo mGluR5 PET
Marker untersucht. [18F]-FTECMO führte zu vielversprechenden in vitro Resultaten,
während die anschliessenden PET Versuche in einer Wistar Ratte unter der rapiden in
vivo Defluorierung litten. Der zweite PET Tracer Kandidat, [18F]-FDEGPECO, verfügte
dann jedoch neben den exzellenten in vitro Eigenschaften auch über gute in vivo
Eigenschaften. In dieser Arbeit wurde klar demonstriert, dass [18F]-FDEGPECO als PET
Tracer für die bildgebende Darstellung von mGluR5 in Ratten verwendet werden kann
und ebenfalls für die Bildgebung in höheren Spezies wie Affen oder Menschen nützlich
sein könnte.
17
Abbreviations
ABP688
3-(6-methyl-pyridin-2-ylethynyl)-cyclohex-2-enone O-methyloxime
AC
adenylat cyclase
ACN
acetonitrile
AMPA
2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)-propionic acid
BBB
blood-brain barrier
Bmax
maximal binding capacity
Bq
Becquerel
BSA
bovine serum albumin
BuLi
butyl lithium
cAMP
cyclic adenosine monophosphate
clogP
calculated logP
CDPPB
3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide
CNS
central nervous system
CPCCOEt
7 - hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl
ester
CPPHA
N- 4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2yl)methyl]phenyl -2-hydroxybenzamide
CT
computer tomography
d
doublet
DAG
diacylglycerol
dd
double doublet
18 DFB
3, 3’-difluorobenzaldazine
DMF
dimethylformamide
Et2O
diethylether
Et3N
triethylamine
EtOAc
ethylacetate
F-PEB
3-fluoro-5-(pyridin-2-ylethynyl)benzonitrile
F-MTEB
3-fluoro-5-((2-methylthiazol-4-yl)ethynyl)benzonitrile
FDG
fluorodeoxyglucose
FPECMO
(E)-3-((6-fluoropyridin-2-yl)ethynyl)cyclohex-2-enone O-methyl
oxime
g
gram
GPCR
G-protein coupled receptor
GABA
γ-aminobutyric acid
h
hours
HEPES
4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid
HPLC
high pressure liquid chromatography
IC50
inhibition constant required for displacement of 50% of radioligand
binding
ID
injected dose
IDnorm./g
injected dose normalized to body weight per tissue weight
KD
dissociation constant
Ki
inhibition constant
LDP
long term depression
LTP
long term potentiation
M
molar
m
multiplett
mg
milligram
mGluR
metabotropic glutamate receptor
mGluR5
metabotropic glutamate receptor of the subtype 5
min
minutes
M-MPEP
2-methyl-6-((methoxyphenyl)ethynyl)-pyridine
MeCN
acetonitrile
MPEP
6-methyl-2-(phenylethynyl)-pyridine
MPEPy
2-((3-methoxyphenyl)ethynyl)pyridine
MTEP
2-methyl-4-(pyridin-3-ylethynyl)thiazole
MRI
magnetic resonance imaging
MS
mass spectroscopy
NMDA
N-methyl-D-aspartate
NMDAR
N-methyl-D-aspartate receptor
NMR
nuclear magnetic resonance
P-gp
P-glycoprotein
PBS
phosphate buffered salin
Pd(PPh3)4
tetrakis(triphenylphosphin)palladium
PEG
polyethyleneglycol
PEI
polyethylenimine
PET
positron emission tomography
19
20 PI
phosphoinositol
PLC
phospholipase C
ROI
region of interest
Rt
room temperature
SAR
structure-activity relationship
SP203
3-fluoro-5-((2-(fluoromethyl)thiazol-4-yl)ethynyl)benzonitrile
SUV
standard uptake value
TAC
time-activity curve
TBAF
tetrabutylammoniumfluoride
TBS
tertiary -butyldimethylsilyl
THF
tetrahydrofuran
TLC
thin-layer chromatography
TM
transmembrane domain
Tris
tris(hydroxymethyl)aminomethane
UV
ultraviolet
Introduction
22
Introduction 1.1
Positron Emission Tomography
Positron Emission Tomography (PET) is a nuclear medicine imaging technique, allowing
for the non-invasive and quantitative imaging of physiologic and pathophysiologic
processes in vivo. A maximal resolution of about 1mm can be achieved with this
technique and it is used for functional imaging. The distribution of specific tracers,
radiolabeled with positron emitting nuclides, and their accumulation in specific organs is
detected and reconstructed in three dimensional pictures. [18F]-FDG is by far the most
common PET tracer. This glucose analogue was first administered to human in 1976 and
its application in tumor and brain imaging pushed the development of the PET technique
and its widespread use enormously. Even today, more than 90% of all PET scans detect
[18F]-FDG distribution and thus tissue metabolic activity. However, PET is by far more
than a medical tool and is widely used in research. Numerous biological targets and
systems are in the center of interest in this research field. Specific PET tracers for labeling
of such targets are potential future diagnostics of various diseases and provide elegant
tools for drug development1; especially for the development of drugs with less sideeffects. A growing demand for specific, non-invasive imaging techniques is mainly
denoted in the field of neuroscience. This clearly contributes to the increasing importance
of PET. The technical possibility of combining PET with high resolution imaging devices
such as CT or MRI supports the growing use, in addition2, 3.
1.1.1 PET Principle
The existence of nuclides with excess of protons leads to spontaneous conversion of a
proton into a neutron resulting in positron emission. This beta decay of such positron
emitters is commonly expressed by the following equation:
→
+
+
The energy released during the decay is shared between the positron and the neutrino and
is different for each isotope. The different maximum energies of the most common
positron emitters are listed in Table 1. Positrons are particles with an electric charge of
+1, a spin of 1 2 and the mass of electrons. They form the antiparticle of an electron. The
kinetic energy of the emitted positron is reduced on its way through the surrounding
23 24
tissue or
o other mattter. As sooon as such a low-energ
gy positron is attractedd by an elecctron
the twoo particles collide
c
andd undergo aannihilation. Due to thhe conservaation of chaarge,
linear momentum,
m
, total enerrgy and the conservaation of thee angular m
momentum this
processs results in two gamma ray photonns. Both gam
mma rays em
mitted in ann angle of neearly
180˚ beear an enerrgy of 511 keV. Gam
mma rays are
a electrom
magnetic raddiation of high
energy and with short
s
wavelengths of few
f
picomeeters. Therefore, relativvely few off the
t
whichh allows forr the externnal detectionn. PET cam
meras
gamma rays are abbsorbed in tissue
used foor coincidennce detectioon consist of
o detector pairs arrannged in oppposite directtions
around the radiattion sourcee. Most deetectors con
ntain scintiillators in which ligh
ht is
reemitteed after ioonizing raddiation is absorbed. The light is then deetected by i.e.
photom
multipliers. Gamma rayys detectedd simultaneeously by an oppositee detector pair
display a line that must
m contaiin the placee of annihilaation und thhus the emittting beta prrobe.
Reconsttruction of the signals leads to thrree-dimensional PET-im
mages withh a resolution up
to 1 mm
m dependingg on the PE
ET system used
u
(Fig. 1). Limiting factors for high resolu
ution
is the inndefinite traavel of the positron inn the tissue before anniihilation, anngulation off the
photon pair due too residual positron
p
mootion and th
he configuraation of thee PET scan
nners
4-6
concernning detectoor size and type.
t
Figure 1. Coinciidence deteection and positron emission toomography after posiitron
annihilaation
Introduction 25 1.1.2 PET Nuclides
Commonly, positron emitting isotopes with short half-lives such as oxygen-15, nitrogen13, carbon-11 and fluorine-18 are used for labeling biomolecules or drugs7, 8. The choice
of the nuclide for each novel tracer is made according to the different properties of the
nuclides (Table 1) such as half-life, formation process, the positron range and the
maximum energy and according to the application of the tracer. Unlike oxygen, nitrogen
or carbon, fluorine is not naturally part of organic biomolecules. Thus, introduction of
fluorine-18 effects the structure of a molecule to a greater extent than the introduction of
other PET nuclides. It changes the physicochemical properties and pharmacology of a
tracer, largely. However, due to its favorable physical properties fluorine-18 is often used
as PET nuclide. Even if in terms of bioisosterism the C-F bond is more similar to the C-O
bond, fluorine-18 is often used to replace hydrogen in biomolecules.9
PET-Nuclide
Nuclear reaction
14
15
13
11
68
F
life
in water10
[MeV]
[min]
[mm]
2.1
8.0
1.19
10.0
5.1
1.72
20.4
3.9
0.96
109.7
2.3
0.64
8.9
1.89
N(p,α)11C
C
18
Maximum energy
O(p,α)13N
N
14
Positron range
N(d,n)15O
O
16
Half-
18
O(p,n)18F* : (F-)
18
O(p,n)18F* : (F2)
20
Ne(d,α)18F : (F2)
66
Zn(α,2n)68Ga
Ga
67.8
*Enriched nuclide as target material
Table 1. Formation process, half-life, maximal linear range in water and maximum energy of
clinically used radionuclides for PET
26
Due to the short half-life of most positron emitters the production and radiosynthesis of
PET tracers is limited to facilities equipped with a cyclotron. The only exception is 18F.
Its physical half life of 110 min allows for transport to other institutions, as well.
Nevertheless, the short half-lives of PET nuclides - even in the case of fluorine-18 require rapid and efficient radiolabeling methods in PET chemistry3.
1.1.3 Modi Operandii for the Synthesis of Carbon-11 and Fluorine-18 Labeled
Tracers
Due to the convenient half-lives of carbon-11 and fluorine-18, most of effort is put into
the development of tracers labeled with one of those isotopes. In general, carbon-11 and
fluorine-18 are produced by initializing of a nuclear reaction which requires the
bombardement of convenient stable isotopes with high energetic protons provided by a
cyclotron.
More than one nuclear reaction is known for the production of carbon-11, but the most
common one is the
14
N(p,α)11C-reaction. For this, a nitrogen gas target containing 0.5%
of oxygen is bombarded.11 For the following radiolabeling the resulting [11C] is reacted
either directly in the target or on-line rapidly to form primary intermediates e. g. [11C]CO2, [11C]-CO, [11C]-HCN or [11C]-CH4. These often have to be transferred into more
reactive secondary intermediates such as [11C]CH3I, [11C]-RCHO, [11C]-COCl2, [11C]RCH2NO2; [11C]-RCH2NO2, that allow for various radiolabeling procedures for a broad
variety of different precursors and for the formation of a large range of structurally
different tracers. In order to form those primary and secondary precursors rapidly,
automated synthesis in modules is often established besides on-line and batch-wise
production.
Two different nuclear reactions are commonly used for direct fluorine-18 production.
Since these two methods result in different chemical forms of [18F] it is necessary to
consider in advance which strategy is the most promising for the development of a
radiotracer and its synthesis. The 20Ne(d,α)18F-reaction leads to the formation of [18F]-F2
and the
18
O(p,n)18F-reaction results in [18F]-fluoride. Since the electrophilic labeling
approach with [18F]-F2 results in products with low specific activities, the use of the
nucleophilic approach with [18F]-F- is first choice. Technically, [18F]-fluoride is produced
by proton irradiation of [18O]-enriched water filled in the target. After the irradiation
[18F]-F- is separated from the target water via trapping on a QMA cartridge. It is
Introduction complexed with Kryptofix 222 (K2.2.2.) in the presence of potassium ions or other
monovalent or alkaline ions such as Cs+ and Rb+. Sufficiently high specific activities can
be obtained with this procedure. In summary, with both mentioned labeling agents, [18F]F2 and [18F]-F-, numerous different modes of radiosynthesis are possible. This allows for
the synthesis of a large variety of fluorine-18 labeled compounds.
1.1.4 Central Nervous System PET Tracers – General Aspects and Requirements
The development of successful PET radiotracers for imaging neuroreceptors is a big
challenge since numerous requirements have to be fulfilled as illustrated by the following.
High affinity and selectivity: The two terms are related to the affinity of a ligand
towards its target. Dependent on the way the value was obtained the KD (dissociation
constant) and the Ki (inhibition constant) express the affinity of a ligand to the target.
Usually, binding affinities in the low nanomolar range are desired. Considering the
selectivity of a radioligand the highest affinity should be given for the actual target and
only low affinity - at least by one order of magnitude lower – is tolerated for any other
binding site. In few cases, a lack of selectivity can be accepted e.g. the actual binding site
and the disfavored one are anatomically separated.
Concentration of target sites: A high ratio of binding site concentration towards
radioligand concentration is a requirement for a valid response to changes in binding site
concentration caused by diseases or drug occupancy during a PET experiment. Therefore
it is important that the binding site concentration (Bmax) exceeds the affinity (KD) of the
radioligand. In addition, this positively affects the target to non-target ratio and the
imaging quality.
Optimal lipophilicity: The lipophilicity of a radioligand for CNS targets plays a certain
role for two reasons. On the one hand, the compounds have to pass the blood-brain barrier
and therefore should have a logP value above 1 to pass the BBB just by diffusion. On the
other hand, non-specific binding is known to be higher with increasing lipophilicity
among structurally related compounds12. Thus, the upper limit for convenient logP values
is estimated to 313.
Toxicity: In most cases radioligands are safe and show few side effects.14 However, toxic
effects should be considered as soon as high amounts of tracers with low specific activity
and low LD50 values are administered.
27 28
In vivo stability: Rapid metabolism is undesired and could lead to the formation of
radioactive metabolites. Unfavorable is the metabolic elimination of the radioactive
nuclide from the radiotracer. The quality of the images is tremendously reduced if those
metabolically formed products are taken up in the target area during acquisition time. In
conclusion, in vivo stability depending on the structure and the labeling position a
compound should be considered during the design of the ligand.
Efficient radiosynthesis: Due to the short half-life of common PET nuclides a rapid and
efficient radiolabeling process is crucial. Typically, establishment of a one-step
radiosynthesis is desired for obtaining high radiochemical yields and high specific
activities.
High specific activity: The ratio between labeled and unlabeled tracer is defined by the
specific activity. Low specific activities result in low quality PET images. In case of low
binding site concentrations - this applies in particular to neuroreceptor systems - a very
high specific activity is required for successful PET imaging.
The pharmacologic investigation of numerous central nervous system disorders and the
development of specific and potent drugs can significantly be facilitated by PET. Mainly
complex receptors systems are responsible for signal transduction in the CNS. Thus,
receptors are often the targets of interest. The development of selective and potent drugs
modulating receptor activity is time-consuming and expensive. Applying PET can save
both, time and money. During the extensive pre-clinical evaluation of novel ligands the
PET technique provides with information about biodistribution, kinetics and the potency
in vivo. Early decision making is a main cost factor in drug development. In addition,
documentation of drug safety and dosage finding can be supported by PET. However, the
costs for the development of the particular PET tracer have to be taken into consideration,
too. Since such selective receptor radioligands can be used to gain functional,
physiological and biochemical information, further purposes of the application can be the
investigation of receptor pathophysiology in general, or the diagnosis of pathologic states
as well as the therapeutic management of certain patients1, 15-17.
Introduction 1.1.5 PET Tracer development
As soon as the radiolabeling of a tracer of interest is established, the pharmacologic
evaluation of the radioligand is pursued. Several in vitro, ex vivo and in vivo techniques
such as saturation binding studies, stability studies, autoradiography, biodistribution and
metabolite studies are under consideration at this stage. PET studies in human are only
recommended after a PET tracer of interest has demonstrated proper characteristics in
animal PET studies. Commonly, mice and rats are utilized for PET studies in preclinical
stages but monkeys and pigs are possible subjects as well. The evaluation in rodents
occupying special small animal PET systems simplified the step from preclinical studies
to clinical studies18. However, species differences have to be considered when comparing
PET results19.
1.1.6 CNS PET tracers in vivo
Wagner et al. performed the first in vivo imaging studies of dopamine receptors in the
human brain in 1983. It was the beginning of a fast growing trend to use specific PET
tracers for the investigation of neurotransmission in the brain. Numerous specific
radiotracers for imaging physiologic and pathophysiologic processes in the brain have
been developed for targeting various dopamine, serotonin, benzodiazepine or opioid
receptors20,
21
. Besides that, certain effort has been made to find a specific tracer for
imaging mGluR5 (metabotropic glutamate receptors of the subtype 5). The first in vivo
experiment with a carbon-11 labeled tracer in human subjects was reported by Ametamey
et al. in 200622. MGluR5 is one of the most interesting targets for PET imaging with
promising future applications in drug development, diagnosis and therapeutical
monitoring.
29 30
1.2
Glutamatte Recepttors
Glutam
mate receptors are transsmembrane receptors activated
a
byy the amino acid glutam
mate
the maain excitatoory neurotraansmitter inn mammalss. After gllutamate is released from
f
synapsees into the synaptic clleft, it bindds to the glu
utamate binnding site oof the recep
ptors
being presented
p
exxtracellularlly. A changge of confirrmation of the
t receptorr protein results
in varioous biochem
mical reactions and inn an intraceellular respponse. Num
merous diffeerent
glutamaate receptorrs with diffe
ferent propeerties are kn
nown. In geeneral, glutaamate recep
ptors
are classsified as ionnotropic and metabotroopic glutam
mate receptorrs (Fig. 2).
Figure 2.
2 Overview
w of glutamatte receptors..
1.2.1 Ioonotropic Glutamate
G
R
Receptors
Most of
o excitatorry neurotrannsmission is mediated
d by ionottropic glutaamate recep
ptors
(iGluRss) which are
a ligand--gated catiion selectiv
ve channells permeabble to sodium,
potassiuum or calciuum ions. Ioonotropic gluutamate recceptors are named
n
afterr three diffeerent
selectivve ligands (Fig.
(
2). Thhese agonissts, NMDA
A (N-methyl-D-aspartaate), AMPA
A (αamino-33-hydroxyl--5-methyl-issoxazol-4-pproprionate)) and kainaate, bind att the glutam
mate
bindingg site and were
w
the first
f
model substancess used for the investiigation of their
t
Introduction corresponding glutamate receptors23-25. In addition, the orphan glutamate receptors,
GluRδ1 and GluRδ2, are considered ionotropic because of their sequence homology.
However, they are not gated by L-glutamate26, 27.
NMDA receptors are special among ligand-gated ion channels. They require two
obligatory co-agonists, glutamate and glycine, binding at the same time to different
binding sites of the receptor. Besides the presynaptic activity postsynaptic membrane
polarization deriving from AMPA, kainate, mGluR5 or other excitatory inputs is
necessary for NMDA receptor activation28. Toxic Ca2+ levels in neurons, which are
responsible for neurodegeneration, are thought to be a consequence of overstimulation of
NMDA receptors by glutamate29-31. Thus, these receptors seem to be involved in several
different neurological impairments such as epilepsy, stroke, trauma, depression,
schizophrenia or Parkinson’s and Alzheimer’s disease32. AMPA receptors are present in
most of excitatory synapses. There they mediate fast excitatory neurotransmission and
recently, are suspected of being involved in neuronal death as well29. Kainate receptors
are generally less well known than other ionotropic glutamate receptors. Their synaptic
function in the CNS is not yet clear. They mediate slow excitatory currents in
hippocampal neurons. Presynaptically, they seem to cause the release of the inhibitory
neurotransmitter GABA33, 34.
1.2.2 Metabotropic Glutamate Receptors
Metabotropic glutamate receptors are typical G-protein coupled receptors (GPCRs) of the
family C with an extracellular domain forming the glutamate binding site, 7
transmembrane domains (TM) and an intracellular domain (Fig. 3). This is necessary for
the activation of intracellular messenger systems after glutamate binding35.
1.2.2.1 Structure of mGluRs
In comparison to other metabotropic receptors the extracellular hydrophilic N-terminal
domain of mGluRs is extraordinarily large. The extracellular domain is necessary for
glutamate binding and is a target for different selective agonists that played a role for
mGluR distinction (Fig. 3). Two globular domains with a hinge region in between are
involved in glutamate or other ligand binding. The ligand is thought to be trapped by a
change of conformation of this special protein formation36-38. Seven lipophilic
31 32
transmembrane domains (7TM) connect the extracellular parts with the intracellular
domain. G-protein coupling was shown to be provided amongst others by the second
intracellular loop (i2) and the amino portion of the carboxy-terminal ending of the fourth
intracellular loop (i4). In addition the intracellular loop 1 and 3 seem to be involved in Gprotein activation39. The intracellular C-terminal is implicated to interact with proteins of
the Homer family
40
and calmodulin41 thus, being involved in regulation of receptor
function.
MPEP
binding site
N-terminal domain with hinge region
glutamate
binding site
extracellular region
cystein rich domain
7TMD
G-protein coupling
C-terminal domain
intracellular region
Figure 3. Schematic illustration of mGluR5 within the cell membrane as a representative for
metabotropic glutamate receptors.
1.2.2.2 Physiology of mGluRs
Eight known different subtypes belong to the family of mGluRs and are classified in
group I-III of mGluRs based on their pharmacological profile, their sequence homology
and signal transduction pathways (Fig. 2). Group I consists of mGluR1 and mGluR5 that
are positively coupled to phospholipase C (PLC) and cause formation of diacylglycerol
(DAG) and phosphoinositide (PI) resulting in an increase of the intracellular Ca2+ level.
Intracellular signaling caused by group II (mGluR2 and mGluR3) and as well by group III
(mGluR4, mGluR6, mGluR7 and mGluR8) is mediated by inhibition of adenylate cyclase
(AC) and hence the cyclic adenosine monophosphate (cAMP) formation42, 43. While both,
group II and group III mGluRs, are implicated in glutamatergic neurotransmission by
presynaptic mechanisms, group I mGluRs modulate glutamate excitability mainly
postsynaptically35, 44.
Introduction 33 1.2.2.3 Expression of mGluRs
In general, mGluRs are widely expressed throughout the central nervous system.
However, subtype specific presence in certain brain regions varies a lot. Group II mGluRs
are mainly found in the forebrain. Group III mGluRs are expressed throughout the CNS.
One exception is mGluR6, which is primarily expressed in the retina45. Interestingly,
group I mGluRs, mGluR1 and mGluR5, show a distinct expression pattern. Their
distribution in the brain is widely complementary since few brain regions with high levels
of both receptor subtypes have been identified. MGluR1 rich brain regions such as the
cerebellum, the ventral pallidum and the substantia nigra display low levels of mGluR5
expression. The cortex, the striatum or the hippocampus are well equipped with mGluR5
and lack in return high concentrations of mGluR135,
46-49
. However, differences of
mGluR5 expression levels were found in different species and even in different rat
strains19, 50.
1.2.2.4 Pathophysiology of mGluRs
MGluRs are known to mediate slow excitatory neurotransmission and to be involved in
synaptic plasticity. They play a certain role in the modulation of neuronal excitability and
synaptic efficacy51. Thus, it is no surprise that mGluRs were shown to be involved in
numerous CNS disorders such as neuropathic pain52, epilepsy53, 54, Parkinson`s disease5456
, drug abuse54,
57
, cognitive disorders54,
55, 58
, anxiety54,
59
and schizophrenia55,
60
.
Remarkably, group I mGluRs are involved in most of the mentioned CNS diseases. For
this reason, both receptors, but in particular mGluR5, have been investigated intensively
as drug targets.
1.2.2.5 MGluR Pharmacology
The discovery of different potent mGluR ligands played an important role for the
classification of the known subtypes and for the investigation of their involvement in
neurologic disorders. Tremendous effort has been spent on the development of selective
compounds. Both competitive and non-competitive substances were developed that act as
orthosteric and allosteric binders respectively. In both cases the ligands cause either
activation or inhibition. Allosteric ligands may cause no effect as well.
Group I receptors: The very first allosteric mGluR targeting was achieved with a
chromen-1-carboxylethyl derivative, 7 - hydroxyiminocyclopropan[b]chromen-1a-
34
carboxylic acid ethyl ester (CPCCOEt, Fig. 4). It was not structurally related to glutamate
and displayed selectivity for mGluR161. Its binding site was localized within the
transmembrane heptahelical domain of mGluR162. Further substances with improved
affinity have been developed that share the same binding pocket63,
64
. Soon after this
achievement the non-competitive antagonists SIB-1757 and SIB18 shown in Figure 5
were identified as selective ligands for mGluR565. Their structural improvement led to a
potent compound named 2-methyl-6-(phenylethynyl)pyridine (MPEP, Fig. 5) with an
IC50 of 37nM66. Unfortunately, this inverse agonist displayed interaction with the
noradrenaline transporter as well and thus further effort lead to the development of
compounds
with
improved
characteristics
such
as
2-methyl-4-(pyridin-3-
ylethynyl)thiazole (MTEP, Fig. 5), 2-((3-methoxyphenyl)ethynyl)-6-methylpyridine (MMPEP, Fig. 5) or 2-((3-methoxyphenyl)ethynyl)pyridine (MPEPy, Fig. 5)67. The binding
site of these allosteric mGluR5 ligands was shown to be located within the 7TM as well.
Three important non-competitive positive modulators of mGluR5 were described only
few years ago. 3, 3’-difluorobenzaldazine (DFB, Fig. 6), N- 4-chloro-2-[(1,3-dioxo-1,3dihydro-2H-isoindol-2-yl)methyl]phenyl -2-hydroxybenzamide (CPPHA, Fig. 6) and [3cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide] (CDPPB, Fig. 6) interact with the
transmembrane domain region as well but are not structurally related to the known
mGluR5 antagonists68-70.
N
OH
O
O
O
(-)-CPCCOEt
Figure 4. Structure of mGluR1 allosteric antagonist, (-)-CPCCOEt.
Introduction 35 OH
N
N
N
N
SIB-1893
SIB-1757
S
N
N
N
MTEP
MPEP
O
O
NH
N
N
N
O
M-MPEP
O
M-PEPy
N
N
H
Cl
fenobam
Figure 5. Structures of mGluR5 allosteric antagonists.
Cl
O
N
N
N
H
OH
F
F
DFB
CPPHA
O
N
O
O
N
CDPPB
N
N
H
N
Figure 6. Structures of mGluR5 allosteric agonists.
Remarkably, SIB-1893 and MPEP also exhibit positive allosteric modulation of
mGluR471. Furthermore, the mGluR5 postive allosteric modulators, DFB and CPPHA,
also display antagonist activity at mGluR4 and mGluR869.
36
Group II receptors: First success was achieved in Eli Lilly with a selective orthosteric
agonist
for
the
group
II
receptors,
aminobicyclo[3.1.0]hexane-2,6-dicarboxylic
mGluR2
acid,
and
named
mGluR3,
(+)-2(Fig.7)72.
LY354740
Surprisingly, this compound even penetrates the blood-brain barrier and thus even entered
into clinical trials for the treatment of anxiety. However, it has been difficult to design
highly selective orthosteric agonists and antagonists for other mGluRs, since the
extracellular glutamate binding site is highly conserved. Furthermore, orthosteric ligands
usually display similar structure as glutamate and are therefore not likely to penetrate the
blood brain barrier by simple diffusion73. Noncompetitive antagonists for group II
mGluRs were found as well. One of the most successful early compounds is 3-(4-Oxo-7phenylethynyl-4,5-dihydro-3H-benzo[b][1,4]diazepin-2-yl)-benzonitrile
(Ro
67-6221,
Fig. 7).
H
N
HO H
OH
O
O
H
H
O
N
N
LY354740
Ro 67-6221
Figure 7. Structures of group II mGluRs agonist, LY354740, and of the negative allosteric
modulator, Ro 67-6221.
Group III receptors: The most important compound among ligands for group III
mGluRs
is
(1aS,7aS,E)-7-(hydroxyimino)-N-phenyl-1,1a,7,7a-
tetrahydrocyclopropa[b]chromene-1a-carboxamide ((-)-PHCCC, Fig. 8), an mGluR4
positive allosteric modulator. A structural analogue is CPCCOEt that is an mGluR1
antagonist. Other group III mGluR ligands include (S)-2-amino-4-phosphonobutanoic
acid
(S-AP4,
Fig.
8),
an
agonist
for
mGluR4,
-6
and
-8
and
(S)-4-
(amino(carboxy)methyl)phthalic acid (S-DCPG, Fig. 8), an agonist for mGluR874.
However, the pharmacological characterization of group III is still poor.
Introduction Figure 8. Structures of the mGluR4 positive allosteric modulator, (-)-PHCCC, of the
mGluR4,-6 and -8 agonist, (S)-AP4 and of the mGluR8 agonist, (S)-DCPG.
The pharmacological profile of group I mGluRs on the contrary has been elaborated
much more in detail. Compared to other mGluRs, mGluR5 has been in the center of
interest. Numerous compounds have been synthesized to determine therapeutic
approaches for the prospective treatment of mGluR5 related diseases.
1.2.3 PET radioligands for labeling mGluR5
Enormous effort has been put into the pharmacological investigation of mGluR5. But
even if mGluR5 involvement in numerous CNS diseases is clearly proven the
mechanisms are largely unknown and still need to be elucidated. In vitro techniques are
useful to support this endeavor. However, the non-invasive in vivo imaging of the
receptors in the brain is more effective, more elegant and smarter. PET imaging as a
functional imaging technique is suitable for visualisation of complex biochemical
processes in vivo. A prerequisite is of course the availability of appropriate PET imaging
agents. MGluR5 PET tracers are in addition useful tools for drug development.
1.2.3.1 Structure of mGluR5 ligands and binding pocket
Most of the available data have been obtained from structure-activity relationship studies,
homology models based on the bovine rhodopsin receptor and on site-directed
mutagenesis of mGluR5. Up to now, a large number of ligands for mGluR5 have been
synthesized and investigated for their affinities. Most of these compounds are derived
from MTEP or M-MPEP, a more effective analogue of MPEP exhibiting non-competitive
antagonist activity. One exception is fenobam (Fig. 5), a Roche compound, that is
structurally different from MPEP75. However, both, MPEP and fenobam, were shown to
37 38
interact with the same binding pocket within the transmembrane domain region of
mGluR5 and display inverse agonist activity. Site directed mutagenesis was used for the
identification of crucial amino acids for [3H]-MPEP and [3H]-M-MPEP binding. Eight
amino acids were determined to be responsible for the high binding affinity and the ligand
was shown to interact with 4 transmembrane domains: TMIII, TMVII, TMV and TMVI66,
76
. Fenobam has shown to interact similarly with the mGluR5 protein. Thus, a ligand
based pharmacophore alignment of both ligands was proved75, 77.
Only recently, the allosteric mGluR5 agonists DFB and CDPPB68-70 were shown to be
effective competitors of [3H]-MPEPy, an MPEP analogue. Presumably, allosteric
potentiators and the allosteric antagonist respectively inverse agonists share overlapping
binding pockets in the transmembrane domain region of mGluR578. In addition, there is
evidence for overlapping binding pockets of mGluR1- and mGluR5-ligands. CPCOOEt
(Fig. 4), an allosteric mGluR1 antagonist, blocked MPEP binding to mGluR5 in
competition experiments. It is hypothesized that the pyridine ring of MPEP and the
benzene ring of CPCCOEt occupy the same binding area between TMIII and TMVII66.
Kulkarni et al.79 presented a simple model (Fig. 9) for the design of novel selective
mGluR5 antagonists. According to that model the binding site for allosteric mGluR5
antagonists consist of two hydrophobic regions that interact each with one aromatic part
of the typical mGluR5 antagonists. These aromatic parts are placed into the two
hydrophobic regions by a linker, often acetylene. Many compounds bearing this general
structural pattern succeeded as high affinity ligands79. Comparing all binding data around
mGluR5 gives the impression of a distinct role of the involved TMs. In particular, TMIII
and TMVII seem to be important for the affinity of mGluR5 ligands while TMV and
TMVI interaction seem to be important for the selectivity of mGluR5 ligands.
MGluR5 PET tracers are compounds that share this pattern as well. They were obtained
in a developmental process starting from MPEP and M-MPEP. MGluR5 PET tracers
therefore bind to the allosteric antagonist binding site of mGluR5.
Introoduction 3 39
Figu
ure 9. Homoology modell of mGluR55, Kulkarni et
e al.79.
1.2.3.2 Importaant mGluR55 PET radiootracers
Twoo radioisotoopes, carbonn-11 and fluuorine-18, were
w used foor the labelinng of mGlu
uR5 PET
traccers. Due to the short physical
p
half-life of 11C,
C current efffort is mainnly focusing on the
development of
o [18F]-labeeled tracers.. Nevertheleess, the firsst success w
was achieved
d with a
[11C]-labeled
C
compound named
n
[11C]--ABP688 (F
Fig. 10).
S
N
[18F]-FPEB
N
[ 11 C
C]-ABP688
18
N
F
[18 F]-MTEB
CN
N
S
11
O
C 3
CH
F
N
18
F
18F
CN
[18 F]-SP20
03
CN
N
N
N
O
[18 F]- FE-DABP688
18F
18F
N
N
O
CH 3
[18 F]-FPE
ECMO
Figu
ure 10. Struuctures of im
mportant mG
GluR5 PET trracers.
It displayed
d
prreferable chharacteristics in mouse, rat and inn human in vivo PET imaging.
i
Thiss compoundd was the first
f
mGluR
R5 PET traccer used inn clinical stuudies and is
i up till
now
w the gold standard22. 3-fluoro-5-(pyridin-2--ylethynyl)bbenzonitrilee ([18F]-FPE
EB, Fig.
10) and 3-fluoro-5-((2-m
methylthiazool-4-yl)ethy
ynyl)benzonnitrile ([18F]]-MTEB, Fig.
F
10)
werre the first fluorine-188 labeled coompounds to
t be publiished. The low radiocchemical
yielld during syynthesis of the compouunds was diisadvantageeous80, 81. A further intteresting
40
18
F-ligand for mGluR5 presented by Simeon et al. was an MTEB related mGluR5 PET
tracer, named [18F]-SP203 (Fig 10). This tracer showed high in vivo defluorination in
monkeys. In humans, however, a reduced bone uptake of 18F-fluoride was observed and it
was possible to generate useful PET images82, 83. Attempts to generate fluorine-18 labeled
ABP688 analogues led only to moderately successful tracers such as [18F]-FE-DABP688
(Fig. 10) or (E)-3-((6-fluoropyridin-2-yl)ethynyl)cyclohex-2-enone O-methyl oxime
([18F]-FPECMO, Fig. 10). The lesson learnt from these studies helped to develop new
strategies towards the development of new fluorine-18 labeled ABP688 derivatives84, 85.
In Table 2 important in vivo characteristics of ABP688 and related mGluR5 PET ligands
are shown.
PET Tracer
[11C]-
Striatal (st)
uptake
(control
conditions)
[%IDnorm./g]
0.91
Striatal (st)
uptake
(blockade
conditions)
[%IDnorm./g]
0.19
Cerebellar
(ce) uptake
(control
conditions)
[%IDnorm./g]
0.13
Cerebellar
(ce) uptake
(blockade
conditions)
[%IDnorm./g]
0.11
Sacrifice
time
point
[min]
St/Ce
ratio
30
6
blockade
with
MMPEP
1 mg/kg
[%]
79.1
0.45
0.23
0.22
0.20
20
2.05
48.9
0.18
0.11
0.08
0.09
30
2.25
38.9
ABP688
[18F]FEDABP688
[18F]FPECMO
Table 2. Biodistribution data of [11C]-ABP688 and fluorine-18 labeled derivatives obtained in
rats.
Introduction 1.3
Aim of the Thesis
Intensive investigations during the last years have shown that metabotropic glutamate
receptors (mGluRs) are involved in numerous neurodegenerative diseases such as M.
Parkinson, M. Alzheimer and M. Huntington as well as other central nervous system
disorders such as neurogenic pain, epilepsy, depression or drug addiction. Due to the
importance of the metabotropic glutamate receptor subtype 5 (mGluR5) as a potential
drugable target for the treatment of these impairments the pharmaceutical research
focuses on the investigation of this receptor. Therefore efficient tools for the in vivo
investigation of mGluR5 are of special interest. Non-invasive imaging techniques such as
positron emission tomography (PET) offer the possibility to visualize and analyze
mGluR5 under various physiological and pathophysiological conditions. Very recently,
our group reported the development and the first successful imaging of mGluR5 in
humans using the carbon-11 labeled PET ligand, [11C]-ABP688. However, the short
physical half-life of 20 min for carbon-11 limits its shipment to nuclear medicine
institutions and thus the widespread use. More advantageous is the use of fluorine-18
labeled compounds for imaging mGluR5. Most importantly, the long physical half-life of
fluorine-18 (t1/2 = 110 min) enables “satellite” distribution to clinical PET centers not
equipped with radiochemistry facilities. Only recently, a number of fluorine-18 labeled
compounds for imaging mGluR5 have been reported. Among these are [18F]F-PEB and
the thiazole derivative [18F]SP203. Both mGluR5 PET tracers are useful but have some
shortcomings. [18F]F-PEB was obtained in low radiochemical yields. [18F]SP203 on the
other hand exhibited unfavorable metabolic profile which resulted in radiodefluorination
and subsequent accumulation of [18F]-fluoride in bones and skull. The aim of this project
is therefore to develop an [18F]-labeled PET radioligand that could be used in vivo to
image mGluR5. The [18F]-labeled compound must be radiosynthetically easily accessible
and metabolically stable. The novel ligands will be derivatives of ABP688 so that they
still contain the main structural motif of this well characterized mGluR5 PET tracer. The
realization of this goal will make possible the investigation of diseases in which the
mGluR5 is implicated in nuclear medicine institutions that lack a radiochemistry
infrastructure.
41 42
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Jaeschke, G.; Porter, R.; Büttelmann, B.; Ceccarelli, S. M.; Guba, W.; Kuhn, B.;
Kolczewski, S.; Huwyler, J.; Mutel, V.; Peters, J.-U.; Ballard, T.; Prinssen, E.;
Vieira, E.; Wichmann, J.; Spooren, W., Synthesis and biological evaluation of
fenobam analogs as mGlu5 receptor antagonists. Bioorg. Med. Chem. Lett. 2007,
17 (5), 1307-1311.
76.
Malherbe, P.; Kratochwil, N.; Zenner, M.-T.; Piussi, J.; Diener, C.; Kratzeisen, C.;
Fischer, C.; Porter, R. H. P., Mutational Analysis and Molecular Modeling of the
Binding Pocket of the Metabotropic Glutamate 5 Receptor Negative Modulator 2Methyl-6-(phenylethynyl)-pyridine. Mol. Pharmacol. 2003, 64 (4), 823-832.
77.
Malherbe, P.; Kratochwil, N.; Mühlemann, A.; Zenner, M.-T.; Fischer, C.; Stahl,
M.; Gerber, P. R.; Jaeschke, G.; Porter, R. H. P., Comparison of the binding
pockets of two chemically unrelated allosteric antagonists of the mGlu5 receptor
and identification of crucial residues involved in the inverse agonism of MPEP. J.
Neurochem. 2006, 98 (2), 601-615.
78.
Chen, Y.; Nong, Y.; Goudet, C.; Hemstapat, K.; de Paulis, T.; Pin, J. P.; Conn, P.
J., Interaction of novel positive allosteric modulators of metabotropic glutamate
receptor 5 with the negative allosteric antagonist site is required for potentiation of
receptor responses. Mol. Pharmacol.2007, 71 (5), 1389-98.
79.
Kulkarni, S. S.; Nightingale, B.; Dersch, C. M.; Rothman, R. B.; Newman, A. H.,
Design and synthesis of noncompetitive metabotropic glutamate receptor subtype
5 antagonists. Bioorg. Med.Chem. Lett. 2006, 16 (13), 3371-5.
50
80.
Terence G. Hamill, S. K., Christine Ryan, Celine Bonnefous, Steve Govek, T. Jon
Seiders, Nicholas D.P. Cosford, Jeffrey Roppe, Ted Kamenecka, Shil Patel,
Raymond E. Gibson, Sandra Sanabria, Kerry Riffel, Waisi Eng, Christopher King,
Xiaoqing Yang, Mitchell D. Green, Stacey S. O'malley, Richard Hargreaves, H.
Donald Burns,, Synthesis, characterization, and first successful monkey imaging
studies of metabotropic glutamate receptor subtype 5 (mGluR5) PET radiotracers.
Synapse 2005, 56 (4), 205-216.
81.
Ji-Quan Wang; Werner Tueckmantel; Aijun Zhu; Daniela Pellegrino; Anna-Liisa
Brownell, Synthesis and preliminary biological evaluation of 3-[18F]fluoro-5-(2pyridinylethynyl)benzonitrile as a PET radiotracer for imaging metabotropic
glutamate receptor subtype 5. Synapse 2007, 61 (12), 951-961.
82.
Siméon, F. G.; Brown, A. K.; Zoghbi, S. S.; Patterson, V. M.; Innis, R. B.; Pike,
V.
W.,
Synthesis
and
Simple
18F-Labeling
of
3-Fluoro-5-(2-(2-
(fluoromethyl)thiazol-4-yl)ethynyl)benzonitrile as a High Affinity Radioligand for
Imaging Monkey Brain Metabotropic Glutamate Subtype-5 Receptors with
Positron Emission Tomography. J. Med. Chem. 2007, 50 (14), 3256-3266.
83.
Brown, A. K.; Kimura, Y.; Zoghbi, S. S.; Simeon, F. G.; Liow, J.-S.; Kreisl, W.
C.; Taku, A.; Fujita, M.; Pike, V. W.; Innis, R. B., Metabotropic Glutamate
Subtype 5 Receptors Are Quantified in the Human Brain with a Novel
Radioligand for PET. J. Nucl. Med. 2008, 49 (12), 2042-2048.
84.
Lucatelli, C.; Honer, M.; Salazar, J.-F.; Ross, T. L.; Schubiger, P. A.; Ametamey,
S. M., Synthesis, radiolabeling, in vitro and in vivo evaluation of [18F]-FPECMO
as a positron emission tomography radioligand for imaging the metabotropic
glutamate receptor subtype 5. Nucl. Med. Biol. 2009, 36 (6), 613-622.
85.
Honer, M.; Stoffel, A.; Kessler, L. J.; Schubiger, P. A.; Ametamey, S. M.,
Radiolabeling and in vitro and in vivo evaluation of [18F]-FE-DABP688 as a PET
radioligand for the metabotropic glutamate receptor subtype 5. Nucl. Med. Biol.
2007, 34 (8), 973-980.
Structure-Activity Relationships of
Fluorinated ABP688 Derivatives and
the Discovery of a High Affinity
Analogue as a Potential Candidate for
Imaging mGluR5 with PET
Author contributions:
Cindy A. Baumann carried out the syntheses including
radiolabeling, binding experiments and wrote the
paper.
Linjing Mu organized the experiments and contributed
to the evaluation of the NMR spectra.
Sinja Johannsen contributed to the binding
experiments.
52
Chapter 2
2.1
Abstract
The metabotropic glutamate receptor subtype 5 (mGluR5) is recognized to be involved in
numerous central nervous system disorders. In an effort to obtain a fluorine-18 labeled
analogue of ABP688 and to elucidate the interaction of ABP688 derivatives with the
receptor, 13 novel ligands based on the core structure of ABP688 were synthesized. The first
series represents molecules in which the position of the nitrogen relative to the acetylenic
linker was shifted. The second and third series of compounds contain fluorobenzonitriles,
fluoropyridines and fluorinated oxygen containing alkyl side chains, respectively, at the
oxime moiety. The binding affinities of the compounds clearly show that substituents at the
oxime functionality are well tolerated. Five candidates exhibited binding affinities below 10
nM. The most promising candidate, (E)-3-(pyridin-2-ylethynyl)cyclohex-2-enone O-2-(2fluoroethoxy)ethyl oxime (38), was radiolabeled with fluorine-18. Scatchard analysis
confirmed the high binding affinity (KD1 = 0.61 ± 0.19 nM and KD2 = 13.73 ± 4.69 nM) of
[18F]-38. These data strongly suggest the further evaluation of [18F]-38 as a PET tracer
candidate for imaging the mGluR5.
2.2
Introduction
The amino acid glutamate is the main excitatory neurotransmitter 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, α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) and N-methyl-Daspartate (NMDA) receptors mediate in general fast excitatory neurotransmission. On the
other hand the 8 metabotropic glutamate receptor subtypes (mGluRs) discovered only from
1991 on, however, are coupled to several second messenger cascades and are involved in the
fine-tuning of neuronal activity. The mGluRs belong to the family of G-protein coupled
receptors (GPCRs) and are 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
53
54
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 glutamate receptors have been implicated in numerous central nervous
system (CNS) disorders. Especially, mGluR5 was shown in cell culture and in several animal
models to play a certain role for the development of neurodegenerative diseases like
Alzheimer's Disease4, 5 and Parkinson`s Disease6, 7 or CNS disorders such as schizophrenia8,
9
, depression, anxiety10, neuropathic pain11,
12
, drug addiction13 and fragile X syndrome14.
Although the underlying pathophysiological 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 site16, the seven transmembrane domains typical for GPCRs and an intracellular Cterminus that interacts with the G-protein. The orthosteric binding site is highly conserved
among glutamate receptors, hampering the development of subtype-specific ligands. Most
effort is, therefore, spent on the development of allosteric modulators with their binding site
located within the seven transmembrane domains of the receptor protein.17 The allosteric
antagonist binding site of mGluR518 was investigated intensively with 2-methyl-6(phenylethynyl)pyridine (MPEP)12,
18
and 2-((3-methoxyphenyl)ethynyl)-6-methylpyridine
(M-MPEP)19, two prototype allosteric antagonists with high binding affinity and selectivity
for mGluR5. Important for MPEP binding to mGluR5 are residues in transmembrane domain
III (TMIII) and TMVII on the one hand and TMIV and TMVI on the other hand.20 Binding to
these transmembrane domains represents the general motif of many mGluR5 antagonists.
Structurally, mGluR5 ligands such as MPEP (1) and M-MPEP (2) or radioligands like [18F]3-fluoro-5-(pyridin-2-ylethynyl)benzonitrile21 (3, [18F]F-PEB, Fig.1), [18F]-3-fluoro-5-((2methylthiazol-4-yl)ethynyl)benzonitrile21 (6, [18F]F-MTEB, Fig.1) and [18F]SP20322,
23
(7,
Fig. 1) 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, [11C] –
ABP688 (4, Fig.1), 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
Chapter 2
55
acetylene linker. Although [11C] –ABP688 is an excellent imaging agent, its use is limited to
PET centers with a cyclotron and radiochemistry facilities, mainly due to the short physical
half life of carbon-11. Consequently, there is a need for a fluorine-18 labeled analogue of
ABP688 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.
H 3C
H 3C
N
N
O 11 CH 3
4, [11C]-ABP688
1, MPEP
H 3C
N
N
18 F
OCH3
N
N
OCH 3
5, [18F]-FPECMO
2, M-MPEP
S
S
N
N
18
6, [18F]F-MTEB
3, [18F]F-PEB
CN
18
F
F
CN
18
F
N
7, [18F]SP203
F
CN
Figure 1. Structures of mGluR5 antagonists and radioligands.
The introduction of a fluorine atom into a molecule changes its structure and therefore affects
properties such as lipophilicity, binding affinity or chemical and metabolic stability.
Recently, our group reported on [18F]-F-PECMO24 (5, Fig.1), the first fluorinated analogue
of ABP688 bearing fluorine-18 at the pyridine functionality. [18F]-F-PECMO exhibits high in
vitro binding affinity towards mGluR5 but shows rapid defluorination in vivo. In an effort to
develop more stable fluorinated analogues we designed and synthesized several new
56
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 ABP688 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.
2.3
Chemistry
In Scheme 1 is shown the synthetic pathway leading to compounds 10 and 11. Alkyne
derivative 9 was obtained as previously described.24 Palladium catalyzed Sonogashira
coupling25 of 9 to 3-bromopyridine (8a) or 4-bromopyridine (8b) gave compounds 10 and 11,
respectively, in reasonable yields. Compounds 16-18, which are key intermediates for the
preparation of a large series of ABP688 analogues, were prepared according to the reaction
pathway outlined in Scheme 2. The reaction of ketone 12 with hydroxylamine hydrochloride
yielded a mixture of trans- and cis-oximes that were easily separated by flash column
chromatography to give pure trans- 13 and cis- 14 in 59% and 9% yield, 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-ethinylcyclohex2-enoneoxime (14) to 15a afforded compound 18.
N
Y
X
a
Y
X
N
+
Br
8a; X = N, Y = CH
8b; X = CH, Y = N
aReagents
OCH 3
9
OCH 3
10; X = N, Y = CH
11; X = CH, Y = N
and conditions: (a) Pd(PPh3) 4, CuI, Et3 N, DMF, rt, 24h.
Scheme 1. Synthetic pathway towards compounds with varying positions of the nitrogen atom in
the pyridine ring (Series 1 compounds)a
Chapter 2
57
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 fluoro-benzonitriles at the
oxime functionality of the ABP688 core structure is depicted in Scheme 3.
N
O
HO
OH
N
a
+
13 (59 %)
12
R
N
Br
b
b
N
15a; R = H
15b; R = CH 3
R
N
14 (9 %)
Br
15a
N
OH
N
16; R = H
17; R = CH3
a
HO
N
18
Reagents and conditions: (a) NH2 OH x HCl, pyridine, rt, 18 h; (b) Pd(PPh 3) 4, CuI, Et 3 N, DMF, rt, 20 h.
Scheme 2. Synthetic pathway towards key intermediates 16 - 18.
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 potentially 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.
58
R2
R2
R
N
N
OH
+
a
R1
N
F
19a; R1 = F, R 2 = H
19b; R 1 = H, R 2 = F
16; (E), R = H
17; (E), R = CH 3
18; (Z), R = H
N
N
R
O
R1
R2
O
F
a
+
F
16; (E)
18; (Z)
R1
20; (E), R = CH 3, R 1 = F, R 2 = H
21; (E), R = H, R 1 = F, R 2 = H
22; (E), R = H, R 1 = H, R 2 = F
23; (Z), R = H, R 1 = F, R 2 = H
CN
N OH
N
N
F
N
N
24
25; (Z), R1 = CN, R2 = H
26; (Z), R1 = H, R 2 = CN
aReagents
27; (E), R1 = CN, R 2 = H
28; (E), R1 = H, R2 = CN
and conditions: (a) DMF, NaH, rt, 2h.
Scheme 3. Synthetic pathway towards fluoropyridines (Series 2 compounds) and
fluorobenzonitriles (Series 3 compounds) a
Series 4 compounds were obtained by the reaction of oxime 16 with oxygen containing
aliphatic starting materials such as 29, 30 and 3326 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 endproduct 31 in moderate yields.
Compound 32 was prepared in a similar way using 1-fluoropropanol (30) as starting material.
For the synthesis of compound 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)26 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 TBAF resulted in the final product 38. Since 38 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 radiosynthesis of
compound [18F]-38, tosylated synthon 36 served as the precursor.
Chapter 2
59
N
N
OH
+ HO
N
N
N
N
n
O
F
O
n
31; n = 1
32; n = 2
29; n = 1
30; n = 2
16
R
a
F
b
N
R
OH
16; R = H
17; R = CH3
N
O
O
OTBS
34; R = H
35; R = CH3
c
R
N
e
N
O
O
R
18F
N
N
O
O
OTos
36; R = H
37; R = CH3
[18F]-38; R = H
d
R
N
N
O
O
F
38; R = H,
39; R = CH3
aReagents
and conditions: (a) DMF, NaH,CH 2Br 2, 40 min, rt; (b) (2-(2-bromoethoxy)ethoxy)(tert butyl)dimethylsilane (33), DMF, NaH, 2 h, rt; (c) 1.THF, TBAF, 1.5 h, rt; 2. CH2 Cl2 , Et 3N, benzenesulf onylchloride,
0°C, 8h; (d) TBAF, THF, 60°C, 5h; (e) K[ 18F]F-Kryptofix 222, DMF, 90o C; 10 min.
Scheme 4. Synthetic pathway towards ABP688 analogues with oxygen containing side chains
(Series 4 compounds).
60
Table 1. In vitro binding data and clogP/logDpH7.4 values of ABP688 analogues.
R1
N R2
α
Compound
number
R1
R2
β
KD [nM]
clogP/
δ
logDpH7.4
10
252 ± 25
1.9
11
54.8 ± 20.2 ε
1.9
40
2.2ζ
1.9
α
ABP688
N
20
21
N
4.4
2.4
(single determination)
β
α
γ
Ki [nM];
1.7 ± 0.2
δ
2.4 ± 0.1
46.8 ± 31.2
3.0
9.15 ± 2.15
2.5
Displacement of [3H]-M-MPEP binding to mGluR5 by test compounds, data are presented as the geometric
mean (n =3)followed by the standard deviation; βScatchard analysis of saturation binding data obtained with
radiolabeled test compound in triplicates; γcalculated logP values (chemDraw), δexperimentally obtained data,
ε
Ki data obtained from displacement experimentsα (n =2); ζ unpublished data
Chapter 2
61
Compound
α
number
β
R1 R2 γ
Ki [nM];
KD [nM]
δ
clogP/
logDpH7.4
22
4.01 ± 0.97 ε
2.5
23
44.04 ± 14.4
2.5
25
49.80 ± 31.15
3.4
28
73.38 ± 28.21
3.8
27
56.10 ± 20.67
3.4
31
8.41 ± 0.64
1.8
32
5.42 ± 0.91
2.0
3.8 ± 0.4
β
38
β
KD1: 0.6 ± 0.2
KD2: 13.7 ± 4.7
2.1
δ
26.8 ± 7.1
39
2.6
δ
N
1.7 ± 0.07
2.1 ± 0.05
62
2.4
Results and Discussion
Chemistry. The total syntheses of all target compounds were accomplished by convergent
syntheses. The intermediates 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 Sonogashira 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 compounds 16, 29 and 30 afforded
compounds 31 and 32 in 31 and 18% yield, 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 ABP688 analogues were
successfully synthesized and investigated for their binding affinity towards mGluR5.
Displacement studies were carried out with each candidate using [3H]-M-MPEP, a well
characterized mGluR5 ligand, and employing rat brain membranes without cerebellum.
ABP688 was used for the determination of nonspecific binding. The resulting inhibition
constants (Ki) of the compounds are listed in Table 1. The prototypical pyridine-3-yl and
pyridine-4-yl ABP688 analogues 10 and 11 with Ki values of 252 ± 25 nM and 54.8 ± 20.2
nM, respectively, were shown to be moderately potent binders with clearly reduced binding
affinity compared to the lead compound ABP688 ( KD = 1.7 nM). The strategy to alter the
position of the pyridine N-atom to stabilize [18F]-FPECMO analogues was thus not further
pursued.
We next explored the possibility of incorporating substitutions 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 ABP688 was conducted. Also, derivatives of ABP688 lacking the methyl group in
the pyridine ring were synthesized. The goal of improving or retaining 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-ABP688 analogues.
From these series, the 3-fluoro substitution in the oxime-linked pyridine ring (R2) exhibited
the highest binding affinity. The corresponding methyl containing compound 20, on the
Chapter 2
contrary, showed a five-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 ABP688 (Table 1) is well tolerated for mGluR5 binding or even
improves mGluR5 binding.27-29 Thus, we hypothesized that the reduction in binding affinity
was related to either the overall size of the methylated analogue 20 or to the increased
lipophilicity. Regarding the 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 affinities < 10 nM have generally logP values between 1.7 and 2.5, whereas compounds
with Ki values ≥ 10 exhibited logP 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 seven-fold decreased affinity could be observed for the methyl containing
derivative 39.
Figure 3. Correlation between (Ki) and clogP values of ligands 20 – 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 ABP688 derivatives to a significantly lower extent than the
shift of the nitrogen in compound 10. Nonetheless, these compounds (20-23, 25-28) did not
show high enough binding affinity to be considered for further evaluation.
63
64
There were no significant differences in the binding affinities 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
ABP688 the trans-isomer is significantly a more potent binder than the cis-isomer.30 The
pegylated desmethyl analogues (31, 32 and 38) showed nanomolar binding affinities with the
longer side chains exhibiting slightly higher affinities. 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 the transmembrane domains TMIII,
TMVII, TMIV and TMVI18. Whether ABP688 and our new ligands bind to exactly the same
domains and if they interact with similar amino acids or not remains unanswered.
Nevertheless, the general structure of successful mGluR5 ligands, as described by Kulkarni
et al.31, 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 ABP688 analogues 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
successfully established (Scheme 4). The conversion of the tosylate precursor 36 into [18F]38 was accomplished using Kryptofix and K2CO3 as base in DMF. The reaction proceeded
for 10 min at 90°C and gave radiochemical yields between 25 and 35% (decay corrected).
Semipreparative purification using acetonitrile and water as mobile phase resulted in high
radiochemical purity (≥ 96 %) of the final product. The specific activity of [18F]-38 was in
the range of 110 – 350 GBq/µmol at the time of quality control.
The shake-flask method32 was used to determine the logDpH7.4 of [18F]-38. As expected from
clogP calculations (Table 1), this ligand displays a moderate lipophilicity with a logD value
of 1.7 ± 0.1 (Table 1, 38). The measured logDpH7.4 suggests that [18F]-38 is sufficiently
Chapter 2
lipophilic for free diffusion across the blood-brain barrier33, although this value is somewhat
lower than the experimentally determined logD value of ABP688. Efforts to increase the
lipophilicity, however, resulted in reduced affinity as shown in Figure 3.
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.
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 independent 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
65
66
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 assuming two different binding
sites. The respective binding parameters 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 4.0 to 15.6 pmol/mg
protein, respectively. Based on these data we can not conclude whether both binding sites are
on mGluR5 or not. The non-linear Scatchard plots could alternatively indicate varying
affinity states of one single binding site due to different receptor states, receptor-effector
coupling or decreasing receptor affinity caused by increasing receptor occupancy. Finally,
artifacts due to technical problems are possible reasons for non-linear Scatchard plots.34
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.35, 36 observed for
a positive allosteric modulator of mGluR5, 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5yl)benzamide (CDPPB), binding to the binding site of negative allosteric modulators of
mGluR5 such as MPEP in parallel. As Chen et al. hypothesize CDPPB acts at overlapping
binding sites in the transmembrane 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 indispensable for a successful PET tracer.
2.5
Conclusion
The synthesis and characterization of 13 novel ABP688 analogues were successfully carried
out. The binding affinities of the 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.
Chapter 2
67
2.6
Experimental Section
General methods. 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 aluminium 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
13
C 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
occurence. Low-resolution 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 reversed phase
column (Gemini 10 µm C18, 300 x 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 determination 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, Kinematica AG) for 1 min at setting 4. The
obtained homogenate was centrifuged (1000 g, 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
pellet (P2), which was resuspended with incubation buffer (5mM Tris/acetate buffer, pH 7.4).
The suspension was again centrifuged (17000g, 20 min, 4 ˚C) followed by resuspension of
68
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
preparation processes. The protein concentration was determined before each experiment by
Bio-Rad Microassay with bovine serum albumin as a standard (Bradford, 1976)37.
Competition binding assays. P2 membranes were incubated with increasing concentrations
of test compound (1 pM – 100 µM), each in triplicate, in incubation buffer II (30 mM
NaHEPES, 110 mM NaCl, 5 mM KCl, 2.5 mM CaCl2xH20, 1.2 mM MgCl2, pH 8). As
radioligand [3H]-M-MPEP (2 nM) was added. Nonspecific binding was determined in the
presence of ABP688 (100 µM). 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. In order to separate free radioligand from the membranes, 4 mL of icecold 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 beta scintillation vials. With a beta
counter (Beckman Instruments, LS 6500, Multi Purpose Scintillation Counter) the
radioactivity retained on the filters was measured. The set of 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).
Saturation assay. P2 membranes (500 µg/mL) were incubated with increasing
concentrations of [18F]-38 (0.25 – 100 nM) each in triplicates in ice-cold incubation buffer II
to give a total volume of 200 µL. Non-specific binding was determined in the presence of
100 µM ABP688 for each concentration of [18F]-38 in triplicates. The test samples were
incubated for 45 min at rt before addition of 4 mL 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 gamma-counter (Wizard, Perkin Elmer). Data analysis was performed for
saturation and Scatchard analysis with Kell-Radlig computer program (McPherson &
Chapter 2
69
Biosoft, Cambridge, UK, 1997). Three independent experiments were carried out with [18F]38 obtained from three independent radiosynthetical productions.
Determination of the lipophilicity of [18F]-38 and of [18F]-39 (logDpH7.4). Radiosynthesis
of [18F]-39 was performed in analogy to the radiolabeling for [18F]-38 (see below). As
described in Wilson et al.32 for the shake flask method, 500 µL 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 5`000 rpm for 3 min was performed to
separate the phases. 50 µL of each phase was pipetted into Eppendorf cups for measurement
of the distribution of the activity with a gamma-counter (Wizard, Perkin Elmer). The
experiment was performed in quintuplicate.
Radiosynthesis of [18F]-38. [18F]-fluoride was produced via the
cyclotron using enriched
18
O-water. For trapping of
18
O(p,n)18F reaction in a
18 -
F 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, K2CO3, 0,5 mg in
MeCN/water (3:1)) was used. The solvents were evaporated at 110ºC under vacuum in the
presence of slight inflow of nitrogen gas. After addition of acetonitrile (1 mL), azeotropic
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 90ºC 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 intelligent pump, a Knauer Variable
Wavelength Monitor UV detector and a Geiger Müller LND 714 counter with Eberlein RM14 instrument using a reversed phase column (Gemini 5µm C18, 250 x 10 mm, Phenomenex)
with a solvent system and gradient as follows: H2O (solvent A), acetonitrile (solvent B); flow
5 mL/min; 0-10 min, B 5%; 10-20 min B 5% → B 50%, 20-50 min B 50%. The fraction
containing the product was collected and the mobile phase was evaporated under vacuum.
The product was finally dissolved in PEG200/water (1:1). Determination of relative
lipophilicity, radiochemical purity and specific activity was carried out during quality control
70
step. Identification of [18F]-38 was achieved by coinjection with reference 38 to the HPLC
system during quality control.
(E)-3-(pyridin-3-ylethynyl)cyclohex-2-enone O-methyl oxime (10). To a carefully degassed
solution of 9 (118.0 mg, 0.79 mmol) and 2-bromopyridine (8a) (164 µl, 1.66 mmol) in Et3N
(12 mL) and DMF (1 mL) was added Pd(PPh3)4 (59 mg, 0.05 mmol) and CuI (24.6 mg, 0.13
mmol). The reaction mixture was stirred at room temperature (rt) for 24 h before sat. NH4Cl
solution (7 mL) was added. The above mixture was then extracted with EtOAc (3 x 20 mL).
The organic layer was washed with water (2 x 20 mL) and brine (20 mL) before it was dried
over Na2SO4 and evaporated to give a brown oil. Purification of the residue by column
chromatography pentane/EtOAc (9:1) gave 10 (45 mg, 38%) as a yellow oil, 99% purity by
HPLC (70% MeCN/H2O over 10 min; tR = 5.94 min). 1H NMR (400 MHz, CDCl3): δ 8.67 (s,
1H), 8.52 (d, J = 5.08 Hz, 1H), 7.72 (d, J = 7.96 Hz, 1H), 7.25 (t, J = 4.88 Hz, 1H), 6.51 (s,
1H), 3.92 (s, 3H), 2.54 (t, J = 7.0 Hz, 2H) 2.37 (t, J = 5.44 Hz, 2 H), 1.81 (quint, J = 6.24, 2
H).
13
C NMR (100 MHz, CDCl3): 155.3, 152.2, 148.7, 138.4, 130.3, 127,3, 123.0, 120.2,
93.4, 89.3, 62.0, 29.5, 22.1, 20.8. MS m/z: 227.0 (M + H)+. HRMS calcd for C14H15N2O,
227.1179; found 227.1172.
(E)-3-(pyridin-4-ylethynyl)cyclohex-2-enone O-methyl oxime (11). This compound was
prepared in analogous way to 10. Reacting 9 (82.7 mg, 0.55 mmol) with 4-bromopyridine
hydrochloride 8b (300.2 mg, 1.9 mmol) in 11 mL of Et3N gave 11 only after performing
column chromatography twice. (16 mg, yield = 13%). 1H NMR: (400 MHz, CDCl3): 8.58 (d,
J = 6.4Hz, 2H), 7.29 (d, J = 6.5 Hz, 2H), 6.54 (s, 1H), 3.93 (s, 3H), 2.55 (t, J = 6.9 Hz, 2H),
2.37 (t, J = 6.5 Hz, 2H), 1.82 (quint, J = 6.3 Hz, 2H). 13C NMR (100 MHz, CDCl3): 155.2,
149.8, 131.2, 127.0, 125.6, 122.9, 99.2, 96.1, 62.0, 29.4, 22.1, 20.7. MS m/z: 227.0 (M + H)+.
HRMS calcd for C14H15N2O, 227.1179; found 227.1174.
(E)-3-ethynylcyclohex-2-enoneoxime (13) and (Z)-3-ethynylcyclohex-2-enoneoxime
(14). To a solution of 12 (4.12 g, 34.33 mmol) in pyridine (70 mL) was added hydroxylamine
hydrochloride (3.56 g, 51.46 mmol). The mixture was stirred at rt for 18 h before water (30
mL) was added. The reaction mixture was then extracted with Et2O and the combined
organic layers were washed with sat. CuSO4 solution. The organic layer was washed with
brine, dried over Na2SO4 and evaporated under reduced pressure. Purification of the crude by
Chapter 2
column chromatography Et2O/pentane (1:9) gave the desired trans product 13 (2.738 g, 59%)
as well as the cis isomer (404 mg, 9%). trans product : 1H NMR (400 MHz, CDCl3): δ 6.50
(s, 1H), 3.87 (s, 1H), 2.59 (t, J = 6.6 Hz, 2H), 2.31 (t, J = 6.7 Hz, 2H), 1.84 (quint, J = 6.7
Hz, 2H). cis product : 1H NMR (400 MHz, CDCl3): δ 7.55 (s, 1 H), 7.15 (s, 1H), 3.21 (s, 1H),
2.40 – 2.33 (m, 4H), 1.86 (quint, J = 6.0 Hz, 2 H). MS m/z: 135.93 (M + H)+.
(E)-3-(pyridin-2-ylethynyl)cyclohex-2-enone oxime (16). A solution 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 (110mg, 0.097 mmol) was added. After stirring 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 room temperature the synthesis was quenched with sat.
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 → 7:3) lead 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 + H)+. HRMS calcd for
C13H12N2O, 212.0950; found 212.0945.
(E)-3-((6-methylpyridin-2-yl)ethynyl)cyclohex-2-enone oxime (17). This compound was
prepared in analogous way to 16. 2-bromo-6-methylpyridine 15b (2.4 g, 13.9 mmol) and 13
(1.8 g, 13.3 mmol) gave a pale yellow solid (2.1 g, yield = 70%). 1H NMR (400 MHz,
CDCl3): δ 7.55 (t, J = 8.6 Hz, 1H), 7.28 (d, J = 7.9 Hz, 1H), 7.10 (d, J = 8.6 Hz, 1H), 6.61 (s,
1H), 2.61 (t, J = 6.6 Hz, 2H), 2.57 (s, 3H), 2.41 (t, J = 5.3 Hz, 2H), 1.83 (quint, J = 7.3 Hz,
2H). 13C NMR (100 MHz, CDCl3): δ 20.8, 21.5, 24.3, 29.3, 90.0, 91.6, 122.8, 124.4, 126.9,
131.8, 136.6, 142.4, 156.1, 159.0. MS m/z 226.97 (M + H)+.
(Z)-3-(pyridin-2-ylethynyl)cyclohex-2-enone oxime (18). This compound was prepared in
analogous way to 16. Starting materials 15a (2.190 g, 13.9 mmol) and 14 (925 mg, 6.85
mmol) gave yellow crystals (2.7 g, yield = 85%). 1H NMR (400 MHz, CDCl3): δ 8.61 (d, J =
5.2 Hz, 1H), 7.68 (t, J = 7.8 Hz, 1H), 7.47 (d, J = 8.4 Hz, 1H), 7.28 (s, 1H) 7.24 (t, J = 5.1
71
72
Hz, 1H), 2.47 (t, J = 6.0 Hz, 2H), 2.42 (t, J = 6.6 Hz, 2H), 1.90 (quint, J = 6.6 Hz, 2 H). 13C
NMR (100 MHz, CDCl3): δ 22.2, 27.5, 30.4, 90.1, 92.4, 122.9, 123.1, 127.3, 130.3, 136.3,
139.6, 142.9, 150.1. MS m/z 212.95 (M + H)+. HRMS calcd for C13H12N2O; 212.0950,
found, 213.0365.
(E)-3-((6-methylpyridin-2-yl)ethynyl)cyclohex-2-enone
O-6-fluoropyridin-2-yl
oxime
(20). To a solution of 17 (200 mg, 0.88 mmol) in dry DMF (20 mL) in a flame dried flask
under argon was added NaH, 60% dispersion, (119 mg, 2.98 mmol). After stirring at rt for 30
min 2,6-difluoropyridine 19a (115 mg, 0.99 mmol) was added. The reaction was stirred for
further 1.5 h before it was quenched with 50% NaHCO3 (20 mL) and extracted with ether.
The combined organic layers were washed with water and brine and the solvents were
evaporated off under reduced pressure. Purification of the crude by column chromatography
pentane/EtOAc (3:1) gave white flakes (130 mg, yield = 45%), 100% purity by HPLC (tR =
8.39 min). 1H NMR (400 MHz, CDCl3): δ 7.78 (q, J = 8.5 Hz, 1H), 7.57 (t, J = 7.8 Hz, 1H),
7.30 (d, J = 8.5 Hz, 1H), 7.16 (d, J = 8.5 Hz, 1H), 7.12 (d, J = 8.5 Hz, 1H), 6.73 (s, 1H), 6.61
(d, J = 8.5 Hz, 1H), 2.84 (t, J = 7.8 Hz, 2H), 2.58 (s, 3H), 2.48 (t, J = 5.4 Hz, 2H), 1.88
(quint, J = 7.0 Hz, 2H).
13
C NMR (100 MHz, CDCl3): δ 20.6, 23.1, 24.6, 29.5, 88.6, 93.5,
102.7 (d, J = 35.1 Hz), 104.9 (d, J = 4.6 Hz), 123.0, 124.6, 129.4, 131.4, 136.4, 142.1, 143.5
(d, J = 7.9 Hz), 159.2, 160.0, 161.9 (d, J = 241.8 Hz), 163.7 (d, J = 14.7 Hz). 19F NMR (376
MHz, CDCl3): δ – 68.76 (d, J = 5.9 Hz). MS m/z 321.97 (M + H)+. HRMS calcd for
C19H17FN3O, 322.1350, found, 322.1358.
(E)-3-(pyridin-2-ylethynyl)cyclohex-2-enone O-6-fluoropyridin-2-yl oxime (21). This
compound was prepared in analogous way to 20. Starting material 16 (203 mg, 0.96 mmol)
and 19a (115 mg, 0.99 mmol) gave white flakes (151mg, yield = 51%), 100% purity by
HPLC (tR = 7.07 min). 1H NMR (400 MHz, CDCl3): δ 8.62 (d, J = 5.1 Hz, 1H), 7.78 (q, J =
8.2 Hz, 1H), 7.69 (t, J = 8.1 Hz, 1H), 7.48 (d, J = 8.4 Hz, 1H), 7.26 (t, J = 7.0 Hz, 1H), 7.16
(d, J = 8.8 Hz, 1H), 6.74 (s, 1H), 6.62 (d, J = 7.8 Hz, 1H), 2.85 (t, J = 6.8 Hz, 2H), 2.49 (t, J
= 5.7 Hz, 2H), 1.90 (quint, J = 6.2 Hz, 2H). 13C-NMR (100 MHz, CDCl3): 20.6, 23.1, 29.5,
89.1, 93.2, 102.7 (t, J = 35.5 Hz), 104.9 (d, J = 5.1 Hz), 123.1, 127.4, 129.6, 131.3, 136.2,
142.9, 143.5 (t, J = 7.9 Hz), 150.2, 159.9, 161.8 (d, J = 217.0 Hz), 163.8 (d, J = 17.4 Hz). 19F
Chapter 2
NMR (375 Hz, CDCl3): δ - 68.7 (d, J = 7.79 Hz). MS m/z: 307.94 (M + H)+. HRMS calcd for
C18H15FN3O; 308.1194, found, 308.1194.
(E)-3-(pyridin-2-ylethynyl)cyclohex-2-enone O-3-fluoropyridin-2-yl oxime (22). This
compound was prepared in analogous way to 20. Starting materials 16 (100 mg, 0.47 mmol)
and 19b (71 mg, 0.61 mmol) with NaH, 60%, (25 mg, 0.61 mmol) gave red crystals (117 mg,
81%), 100% purity by HPLC (tR = 4.95 min). 1H NMR (400 MHz, CDCl3): δ 8.59 (t, J = 5.0
Hz, 1H), 8.07 (d, J = 4.8 Hz, 1H), 7.66 (t, J = 7.7 Hz, 1H), 7.47 – 7.37 (m, 2H), 7.24-7.20 (m,
1H), 7.03 – 6.99 (m, 1H), 6.83 (s, 1H), 2.87 (t, J = 6.5 Hz, 2H), 2.47(t, J = 6.0 Hz, 2H), 1.94
– 1.89 (m, 2H). 13C NMR (100 MHz, CDCl3): δ 20.7, 23.2, 29.6, 89.1, 93.1, 119.4 (d, J = 1.7
Hz), 123.1, 124.3 (d, J = 15.6 Hz), 127.5, 129.6, 131.2, 136.2, 142.4 (d, J = 6.42 Hz), 143.0,
146.6 (d, J = 259.3 Hz), 150.2, 152.7 (d, J = 10.3 Hz), 161.2. 19F NMR (375 Hz, CDCl3): δ –
137.34 (t, J = 7.19 Hz). MS m/z 307.94 (M + H)+. HRMS calcd for C18H15FN3O, 308.1194;
found, 308.1205.
(Z)-3-(pyridin-2-ylethynyl)cyclohex-2-enone O-6-fluoropyridin-2-yl oxime (23). This
compound was prepared in analogous way to 22. Compound 18 (190 mg, 0.9 mmol) and 19a
(150 µl, 1.64 mmol) yielded brown crystals (157 mg, 57%), 98% purity by HPLC ( tR = 6.99
min). 1H NMR (400 MHz, CDCl3): δ 8.61 (d, J = 5.2 Hz, 1H), 7.78 – 7,67 (m, 2H), 7.48 (d, J
= 8.0 Hz, 1H), 7.42 (s, 1H), 7.26 (t, J = 5.7 Hz, 1H), 7.08 (d, J = 8.3 Hz, 1H), 6.58 (d, J = 8.4
Hz 1H), 2.58 (t, J = 6.6 Hz, 2H), 2.52 (t, J = 6.2 Hz, 2H), 1.97 (quint, J = 6.3 Hz, 2H). 13C
NMR (100 MHz, CDCl3): δ 22.1, 27.4, 30.5, 88.9, 94.2, 102.3 (d, J = 35.2 Hz), 104.8 (d, J =
5.1 Hz), 123.0, 123.3, 127.6, 134.2, 136.3, 142.7, 143.4 (d, J = 8.2 Hz), 150.3, 156.7, 162.1
(d, J = 242.0 Hz), 163.6 (d, J = 14.4 Hz). 19F NMR (CDCl3): δ – 68.64 (d, J = 3.31 Hz). MS
m/z: 308.04 (M + H)+. HRMS calcd for C18H15FN3O, 308.1194; found, 308.1185.
(Z)-4-fluoro-2-(3-(pyridin-2-ylethynyl)cyclohex-2-enylideneaminooxy)benzonitrile (25)
and
(Z)-2-fluoro-4-(3-(pyridin-2-ylethynyl)cyclohex-2-enylideneaminooxy)benzonitrile
(26). These compounds were prepared in analogous way to 20. Starting materials 18 (203
mg, 0.96 mmol) and 24 (184 mg, 1.32 mmol) gave white crystals 25, (60 mg, yield = 18%),
96% purity by HPLC (tR = 9.9 min), and white crystals 26 (14 mg, 5%). 25: 1H NMR (400
MHz, CDCl3): δ 8.63 (d, J = 3.9 Hz, 1H), 7.70 (t, J = 5.9 Hz, 1H), 7.47-7.57 (m, 2H), 7.35
(d, J = 8.0 Hz, 1H), 6.72-6.80 (m, 2H), 6.97 (s, 1H), 2.89 (t, J = 5.1 Hz, 2H), 2.49 (t, J = 4.5
73
74
Hz, 2H), 1.91 (m, 2H).
13
C NMR (100 MHz, CDCl3): δ 20.6, 23.0, 29.5, 88.9, 93.5, 99.1,
102.9 (d, J = 24.3 Hz), 110.7 (d, J = 7.9 Hz), 114.4, 123.3, 127.4, 129.1, 131.8, 133.2 (d, J =
219.1 Hz), 136.2, 138.2, 141.7, 150.3, 160.1, 165.7. 19F NMR (376 MHz, CDCl3) : δ - 67.77.
MS m/z 331.91 (M + H)+, HRMS calcd for C20H15FN3O, 332.1194; found, 332.1195. 26: 1H
NMR (400 MHz, CDCl3): δ 8.63 (d, J = 3.5 Hz, 1H), 7.70 (t, J = 5.7 Hz, 1H), 7.48-7.57 (m,
2H), 7.35 (d, J = 7.9 Hz, 1H), 6.78 (d, J = 7.9 Hz, 1H), 6.73 (d, J = 6.5 Hz, 1H), 6.97 (s, 1H),
2.89 (t, J = 5.0 Hz, 2H), 2.49 (t, J = 4.6 Hz, 2H),1.91 (m, 2H). MS m/z 331.91 (M + H)+.
(E)-4-fluoro-2-(3-(pyridin-2-ylethynyl)cyclohex-2-enylideneaminooxy)benzonitrile (27)
and
(E)-2-fluoro-4-(3-(pyridin-2-ylethynyl)cyclohex-2-enylideneaminooxy)benzonitrile
(28). The compounds were prepared in analogous way to 20. Starting materials 16 (200 mg,
0.94 mmol) and 24 (180 mg, 1.29 mmol) gave white crystals, 27 (87 mg, yield = 28%), 98%
purity by HPLC (tR = 10.73 min) and white crystals 28, (110 mg, 35%), 97% purity by HPLC
(tR = 10.59 min). 27: 1H NMR (400 MHz, CDCl3): δ 8.63 (d, J = 4.0 Hz, 1H), 7.69 (t, J = 5.8
Hz, 1H), 7.47-7.5 (m, 2H), 7.26 (m, 1H), 7.14 (dd, J = 2.4, 2.4 Hz, 1H), 7.04 (dd, J = 2.4, 2.1
Hz, 1H), 6.69 (s, 1H), 2.78 (t, J = 6.2 Hz, 2H), 2.49 (t, J = 6.0 Hz, 2H), 1.90 (quint, J = 5.2
Hz, 2H). 13C NMR (100 MHz, CDCl3): δ 20.8, 23.3, 29.5, 88.8, 93.7, 99.2, 101.0, 102.9 (d, J
= 24.9 Hz), 111.5, 114.7, 123.6, 127.3, 129.3, 133.0 (d, J = 226.5 Hz), 136.4, 138.2, 142.7,
150.5, 160.3, 164.0 (d, J = 12.6 Hz). 19F NMR (376 MHz, CDCl3) : δ -99.52. MS m/z 331.97
(M + H)+.HRMS calcd for C20H15FN3O, 332.1194; found, 332.1193. 28: 1H NMR (400 MHz,
CDCl3): δ 8.63 (d, J = 3.9 Hz, 1H), 7.67 (t, J = 5.8 Hz, 1H), 7.53 (dd, J = 6.3, 5.6 Hz, 1H),
7.47 (d, J = 8.0 Hz, 1H), 7.33 (dd, J = 2.9, 2.6 Hz, 1H), 7.25 (t, J = 4.9 Hz, 1H), 6.80 - 6.73
(m, 1H), 6.69 (s, 1H), 2.88 (t, J = 7.5 Hz, 2H), 2.49 (t, J = 6.3 Hz, 2H), 1.90 (quint, J = 6.8
Hz, 2H). 13C NMR (100 MHz, CDCl3): δ 20.6, 23.1, 29.5, 88.9, 93.7, 95.6, 103.1 (d, J = 26.0
Hz), 109.8 (d, J = 21.7 Hz), 115.3, 123.3, 127.4, 128.9, 132.2, 134.6 (d, J = 10.9 Hz), 136.3,
142.8, 150.3, 160.8, 162.7 (d, J = 13.0 Hz) 166.3 (d, J = 253.9 Hz).
19
F NMR (376 MHz,
CDCl3): δ -103.91. MS m/z 331.97 (M + H)+. HRMS calcd for C20H15FN3O, 332.1194;
found, 332.1195.
(E)-3-(pyridin-2-ylethynyl)cyclohex-2-enone O-(2-fluoroethoxy)methyl oxime (31). To a
solution of 16 (110 mg, 0.52 mmol) and 2-fluoroethanol 29 (50 µL, 0.85 mmol) in DMF (35
mL) was added 60% NaH (130 mg, 3.25 mmol). After stirring for 30 min at rt,
Chapter 2
75
dibromomethane (100 mg, 0.58 mmol) was added while color changed quickly from yellow
to dark brown. After 10 min 50% NaHCO3 solution (20 mL) was added and after further 5
min the reaction mixture was extracted with ether (3 x 20 mL). The combined organic layers
were washed with water (2 x 20 mL) and brine (10 mL) and dried over Na2SO4 before the
solvent was evaporated under reduced pressure. The obtained crude reaction mixture was
purified by column chromatography over silica using pentane/ether (1:6), which gave a
slightly yellow oil as pure product (47mg, yield = 31%), 97% purity by HPLC (70%
MeCN/H2O over 10 min; tR = 4.89 min). 1H NMR (400 MHz, CDCl3): δ 8.59 (d, J = 3.6 Hz,
1H), 7.65 (t, J = 5.9 Hz, 1H), 7.44 (d, J = 5.9 Hz, 1H), 7.22 (t, J = 6.4 Hz, 1H), 6.60 (s, 1H),
5.25 (s, 2H), 4.62 (t, J = 4.8 Hz, 1H), 4.50 (t, J = 4.0 Hz, 1H), 3.92 (t, J = 3.7 Hz, 1H), 3.85
(t, J = 3.9 Hz, 1H), 2.59 (t, J = 6.8 Hz, 2H), 2.41 (t, J = 6.4 Hz, 2H), 1.81 (quint, J = 6.8 Hz,
2H).
13
C NMR (100 MHz, CDCl3): δ 20.7, 22.4, 29.4, 68.2 (d, J = 20.2Hz), 82.7 (d, J =
171.1 Hz), 89.7, 92.6, 97.8, 122.9, 127.3, 128.4, 130.7, 136.1, 143.1, 150.1, 157.0. 19F NMR
(376 MHz, CDCl3) : δ -223.93 (m, J = 20.44 Hz). MS m/z 288.89 (M + H)+ HRMS calcd for
C16H18FN2O2, 289.1347; found, 289.1347.
(E)-3-(pyridin-2-ylethynyl)cyclohex-2-enone O-(3-fluoropropoxy)methyl oxime (32).
This compound was prepared in analogous way to 31. Starting from 16 (114 mg, 0.54 mmol)
and 2-fluoropropanol 30 (45 µl, 0.85 mmol) afforded the desired product (29 mg, yield = 20
%), 96.8% purity by HPLC (70% MeCN/H2O over 10 min; tR = 5.61 min). 1H NMR (400
MHz, CDCl3): δ 8.59 (d, J = 6.0 Hz, 1H), 7.65 (t, J = 7.8 Hz, 1H), 7.44 (d, J = 8.4 Hz, 1H),
7.22 (t, J = 6.2 Hz, 1H), 6.61 (s, 1H), 5.20 (s, 2H), 4.59 (t, J = 6.0 Hz, 1H), 4.47 (t, J = 6.0
Hz, 1H), 3.75 (t, J = 6.6 Hz, 2H), 2.59 (t, J = 6.6 Hz, 2H), 2.41 (t, J = 6.3 Hz, 2H), 2.00 (m,
1H), 1.94 (m, 1H), 1.81 (quint, J = 5.8 Hz, 2H). 13C NMR (100 MHz, CDCl3): δ 20.7, 22.4,
29.4, 30.8 (q, J = 20.0 Hz), 64.7, 81.0 (d, J = 172.8 Hz), 89.6, 92.0, 97.6, 122.9, 127.3, 128.2,
130.8, 136.2, 143.1, 150.0, 156.8.
19
F NMR (376 MHz, CDCl3) : δ -222.27 (m, J = 22.18
+
Hz). MS: m/z 303.01 (M + H) HRMS calcd for C17H20FN2O2, 303.1503; found, 303.1501.
(E)-3-(pyridin-2-ylethynyl)cyclohex-2-enone
O-2-(2-(tert-
butyldimethylsilyloxy)ethoxy)ethyl oxime (34). To a solution of trans-3[(pyridine-2yl)ethynyl]cyclohex-2-enoneoxime 16 (151 mg, 0.71 mmol) in DMF (15 mL) was added
60% NaH (37 mg, 0.92 mmol). After stirring for 30 min at rt 2-(2-bromoethoxy)(t-
76
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 further 5
min the reaction mixture was extracted with ethylacetate (3 x 20 mL). The combined organic
layers were washed with water (2 x 20 mL) and brine and dried over Na2SO4 before the
solvent was evaporated under reduced pressure. The obtained crude product was purified by
column chromatography over silica using pentane/EtOAc (6:4), which gave a yellow oil
(253mg, 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 = 6.4 Hz, 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 + H)+. HRMS calcd for
C23H34N2NaO3Si+, 437.2231; found, 437.2224.
(E)-3-((6-methylpyridin-2-yl)ethynyl)cyclohex-2-enone
O-2-(2-(tert-
butyldimethylsilyloxy)ethoxy)ethyl oxime (35). This compound was prepared in 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): 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 + H)+.
(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 THF (12.5
mL) was added TBAF 1M (195 µL). After stirring the mixture for one hour, water (10 mL)
was added. The solution was extracted with EtOAc (3 x 20 mL) and the organic layers were
washed with water (2 x 20 mL) and brine (30 mL) before drying over Na2SO4. The brown
crude that 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-
Chapter 2
77
oxyethyl)oxyethyloxime (crude: 85 mg) was dissolved in dry CH2Cl2 (2 mL). After addition
of Et3N (118 µL) the mixture was set to 0°C. Finally, benzenesulfonylchloride (114.39 mg,
0.6 mmol) was added and after 8 h the mixture was diluted with water. Extraction with ether
(3 x 20 mL) and washing the organic layers with water (2 x 20 mL) and brine (30 mL) before
drying over Na2SO4 lead after evaporation to 176 mg of crude product. Purification by
column chromatography using ether/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).
13
C 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 (M + H).
HRMS calcd for C24H27N2O5S+, 455.1635; found, 455.1631.
(E)-2-(2-(3-((6-methylpyridin-2-yl)ethynyl)cyclohex-2-enylideneaminooxy)ethoxy)ethyl
4-methylbenzenesulfonate (37). This compound was prepared in analogous way to 36.
Starting from 35 (350 mg, 0.82 mmol) yielded a clear oil. (187 mg, yield = 45%). 1H NMR
(400 MHz, CDCl3): δ 7.80 (d, J = 8.1 Hz, 2H), 7.55 (t, J = 8.2 Hz, 1H), 7.34 (d, J = 8.6 Hz,
2H), 7.27 (d, J = 8.1 Hz, 1H), 7.10 (d, J = 7.6 Hz, 1H), 6.55 (s, 1H), 4.25 (t, J = 4.9 Hz, 2H),
3.80 – 3.74 (m, 4H), 3.57 (t, J = 5.1 Hz, 2H), 2.59 – 2.51 (m, 5H), 2.45 (s, 3H), 2.38 (t, J =
6.2 Hz, 2H), 1.79 (quint, J = 6.2 Hz, 2H). 13C NMR (100 MHz, CDCl3): δ 20.8, 21.6, 22.3,
24.5, 29.4, 68.7, 69.1, 69.8, 73.4, 92.0, 99.1, 122.7, 124.5, 127.6, 128.0, 129.8, 130.8, 133.1,
136.4, 142.4, 144.8, 155.8, 159.0. MS m/z 469.47 (M + H).
3-[pyridine-2-yl)ethynyl]cyclohex-2-enone-O-fluoroethyloxyethyloxime (38). TBAF x 3
H2O (227 mg, 0.71 mmol) was dried on a high vacuum system at 45°C for 24 h and was then
dissolved in dry THF (5 mL). A solution of 36 (130mg, 0.29 mmol) in dry THF (12 mL) was
added and after stirring the mixture for five h at 60°C, water (10 mL) was added. The
solution was extracted with ether and the organic layers were washed with water (2 x 20 mL)
and brine (1 x 20 mL) before drying over Na2SO4. The brown crude, that 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
78
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 = 4.2 Hz, 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 (M + H). HRMS calcd for C17H20FN2O2, 303.1503; found,
303.1498.
(E)-3-((6-methylpyridin-2-yl)ethynyl)cyclohex-2-enone O-2-(2-fluoroethoxy)ethyl oxime
(39). This compound was prepared in analogous way to 38. Starting material 37 (170 mg,
0.36 mmol) gave a pale yellow oil (68 mg, 59% yield), 97% purity by HPLC (tR = 5.7 min).
1
H NMR (400 MHz, CDCl3): δ 7.53 (t, J = 7.8 Hz, 1H), 7.26 (d, J = 7.6 Hz, 1H), 7.08 (d, J =
7.7 Hz, 1H), 6.55 (s, 1H), 4.62, (t, J = 4.0 Hz, 1H), 4.50 (t, J = 4.3 Hz, 1H), 4.27 (t, J = 4.7
Hz, 2H), 3.80 – 3.76 (m, 3H), 3.71 (t, J = 4.5 Hz, 1H), 2.58 – 2.55 (m, 5H), 2.39 (t, J = 6.3
Hz, 2H), 1.78 (quint, J = 6.3 Hz, 2H). 13C NMR (100 MHz, CDCl3): δ 20.8, 22.3, 24.6, 29.4,
69.8, 70.4 (d, J = 19.8 Hz), 73.5, 83.1 (d, J = 174.7 Hz), 89.4, 92.1, 122.7, 124.5, 127.5,
130.8, 136.3, 142.6, 155.8, 159.0. 19F NMR (376 MHz, CDCl3): δ -223.10 (m, J = 16.83 Hz).
MS m/z 316.97 (M + H). HRMS calcd for C18H22FN2O2, 317.1660; found, 317.1672.
Acknowledgment. We acknowledge the technical support of Claudia Keller, Mathias Nobst,
Lukas Dialer and Phoebe Lam. We thank Christophe Lucatelli, Evgeny Prusov and StefanieDorothea Krämer for fruitful discussion.
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Auberson, Y. P.; Gasparini, F.; Schubiger, P. A., Radiosynthesis and Preclinical
Evaluation of 11C-ABP688 as a Probe for Imaging the Metabotropic Glutamate
Receptor Subtype 5. J. Nucl. Med. 2006, 47 (4), 698-705.
31.
Kulkarni, S. S.; Nightingale, B.; Dersch, C. M.; Rothman, R. B.; Newman, A. H.,
Design and synthesis of noncompetitive metabotropic glutamate receptor subtype 5
antagonists. Bioorg. Med. Chem. Lett. 2006, 16 (13), 3371-3375.
32.
Wilson, A. A.; Jin, L.; Garcia, A.; DaSilva, J. N.; Houle, S., An admonition when
measuring the lipophilicity of radiotracers using counting techniques. Appl. Radiat.
Isot. 2001, 54 (2), 203-208.
33.
Dischino, D. D.; Welch, M. J.; Kilbourn, M. R.; Raichle, M. E., Relationship Between
Lipophilicity and Brain Extraction of C-11-Labeled Radiopharmaceuticals. J. Nucl.
Med. 1983, 24 (11), 1030-1038.
Chapter 2
34.
Limbird, L. E., Cell Surface Receptors A Short Course on Theory and Methods. 3rd
ed.; Springer: 2005; p 219.
35.
Chen, Y.; Nong, Y.; Goudet, C.; Hemstapat, K.; de Paulis, T.; Pin, J.-P.; Conn, P. J.,
Interaction of Novel Positive Allosteric Modulators of Metabotropic Glutamate
Receptor 5 with the Negative Allosteric Antagonist Site Is Required for Potentiation
of Receptor Responses. Mol. Pharmacol. 2007, 71 (5), 1389-1398.
36.
Kinney, G. G.; O'Brien, J. A.; Lemaire, W.; Burno, M.; Bickel, D. J.; Clements, M.
K.; Chen, T.-B.; Wisnoski, D. D.; Lindsley, C. W.; Tiller, P. R.; Smith, S.; Jacobson,
M. A.; Sur, C.; Duggan, M. E.; Pettibone, D. J.; Conn, P. J.; Williams, D. L., Jr., A
Novel Selective Positive Allosteric Modulator of Metabotropic Glutamate Receptor
Subtype 5 Has in Vivo Activity and Antipsychotic-Like Effects in Rat Behavioral
Models. J. Pharmacol. Exp. Ther. 2005, 313 (1), 199-206.
37.
Bradford, M. M., Rapid and Sensitive Method for Quantitation of Microgram
Quantities of Protein Utilizing Principle of Protein-Dye Binding. Anal. Biochem.
1976, 72 (1-2), 248-254.
83
84
Syntheses and Pharmacological
Characterization of Novel Thiazole
Derivatives as Potential MGluR5
PET Ligands
Author contributions:
Cindy A. Baumann carried out the syntheses
including radiolabeling, in vitro evaluation and wrote
the paper.
Linjing Mu supported the radiosyntheses and the
organic syntheses.
Nicole Wertli was involved in the precursor synthesis
and the pharmacological evaluation of the 18FFTECMO.
Michael Honer carried out the in vivo studies and
evaluated the PET data.
86
Chapter 3
3.1
87
Abstract
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 (Ki = 5.5 ± 1.1 nM), was labeled
with fluorine-18. The radionuclide was incorporated via nucleophilic substitution on a
bromo precursor in acetonitrile at 90°C in a single step to afford the final product in 45%
radiochemical yield and specific radioactivity of 120 GBq/µmol. [18F]-FTECMO
displayed optimal lipophilicity (logDpH7.4 = 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 heterogeneous and displaceable uptake into mGluR5 rich
brain regions such as striatum, hippocampus and cortex, while the cerebellum a brain
region with negligible mGluR5 expression exhibited low radioactive uptake. In vivo PET
studies were planned subsequently in order to investigate the in vivo distribution pattern
of [18F]-FTECMO. The PET imaging with [18F]-FTECMO in a Wistar rat, however,
revealed a low brain uptake. Radioactive uptake into the skull was also observed
suggesting in vivo defluorination. Thus, although [18F]-FTECMO is an excellent ligand
for detection of mGluR5 in vitro, its in vivo characteristics are not optimal for the imaging
of mGluR5 in rats in vivo.
3. 2
Introduction
Glutamate is the predominant excitatory neurotransmitter in the mammalian central
nervous system (CNS). Glutamate receptors form a large family which can be classified
into ionotropic and the metabotropic glutamate receptors. The ligand-gated, cationselective ion channels that form the ionotropic glutamate receptors (iGluRs) mediate fast
excitatory neurotransmission. IGluRs are named kainate, α-amino-3-hydroxy-5-methyl-4isoxazoleproprionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors.
Metabotropic glutamate receptors (mGluRs) are known to be involved in the modulation
of iGluRs and seem to fine-tune neuronal activity. The subclass of mGluRs consists of
eight G-protein coupled receptors (GPCRs) that are sub-divided according to their
receptor pharmacology, amino acid sequence and their secondary messenger systems into
88
three groups (group I –III). Group I comprises mGluR1 and mGluR5 that are mainly postsynaptic receptors activating 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
MGluRs have been implicated in numerous CNS disorders. Notably, mGluR5, which is
predominantly located in the hippocampus, striatum and cortex3, was shown to be
involved in neurodegenerative diseases such as Alzheimer`s disease4, 5, Parkinson`s
disease6, 7 or other disorders such as depression8, anxiety9, schizophrenia10, 11, neuropathic
pain12, 13, drug addiction14 and fragile X syndrome15. However, the role of mGluR5 is not
yet well-understood and it is generally agreed that a better understanding of the receptor
is needed for the development of possible diagnostic tools and effective drugs.16
Positron emission tomography (PET) is a non-invasive in vivo imaging technique that
offers the possibility to visualize and analyze mGluR5 expression under various
physiological and phathophysiological conditions. Our group reported the first successful
mGluR5 PET imaging in rodents and humans with the PET tracer [11C]-ABP688 (Fig. 1,
4). This radioligand, labeled with carbon-11 (t1/2 = 20 min) displayed specific uptake into
mGluR5-rich brain regions in humans17. However, the short physical half-life of carbon11 limits its widespread use. More advantageous seems the use of fluorine-18 (t1/2 = 110
min) labeled compounds for imaging mGluR5. Only recently, a number of fluorine-18
labeled compounds for imaging mGluR5 have been reported. Among these are [18F]FPEB18,
19
(Fig. 1, 1) and the thiazole derivatives [18F]F-MTEB18 (Fig. 1, 2) and
[18F]SP203 (Fig. 1, 3). For the radiosyntheses of [18F]F-PEB and [18F]F-MTEB
microwave heating was applied but both compounds were obtained in low radiochemical
yields. The low radiochemical yields were later on improved when thermal heating was
employed20. [18F]F-PEB18, 19 was recently used for in vivo imaging of mGluR5 in human
studies21. In 2007, Siméon et al. presented [18F]SP20322 (Fig. 1, 3), a fluoromethyl
analogue of F-MTEB, where in vivo radiodefluorination was observed in PET studies
involving monkeys. PET studies in humans, however, showed low uptake of activity into
the skull suggesting a lower radiodefluorination rate in humans23. We recently published
three novel fluorine-18 labeled analogues of ABP688: [18F]-FPECMO24 (Fig. 1, 5), [18F]FE-DABP68825 (Fig. 1, 6) and (E)-3-(pyridin-2-ylethynyl)cyclohex-2-enone O-2-(2[18F]-fluoroethoxy)ethyl
radiodefluorination
in
oxime
vivo
(Fig,
while
1,
7)26.
[18F]-FPECMO
[18F]-FE-DABP688
displayed
underwent
unfavorable
Chapter 3
89
pharmacokinetics. In vitro studies with 7 revealed very high binding affinity of the ligand
to mGluR5 with KD1 of 0.61 ± 0.19 nM occupying rat brain homogenate without
cerebellum. Further in vitro and in vivo evaluation of the tracer is currently under way. To
date, [11C]-ABP688 and [18F]SP203 are the most successful and well characterized PET
tracers for the imaging of mGluR5 in humans. Since the introduction of fluorine on the
pyridine ring of ABP688 resulted in defluorination of [18F]-FPECMO, the incorporation
of fluorine into other positions on the pyridine ring was not further pursued. Instead, we
sought to derivatize ABP688 via a thiazole moiety. Herein, we report the syntheses and
binding affinities of 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.
N
18F
1 , [ 18 F]F-PEB
N
N
O11CH3
4, [11C]-ABP688
CN
S
N
18
18
2, [18 F]F-MTEB
N
F
F
N
OCH 3
5, [ 18F]-FPECMO
CN
S
18
F
N
N
F
N
18
O
F
3, [ 18F]SP203
CN
6, [ 18 F]-FE-DABP688
N
N
O
O
7 , (E)-3-(pyridin-2-ylethynyl)cyclohex-2enone O-2-(2-[18F]-f luoroethoxy)ethyl
oxime
Figure 1. Structures of mGluR5 radioligands.
18
F
90
S
H3 CO
O
Si
S
N
N
F
a
10
Br
Br
F
N
OCH 3
b
11
9
8
N
Reagents and conditions: (a) NH2 OCH3 x HCl, pyridine, RT, 18h, 63%; (b) CuI,
Pd(PPh3 )4 , DMF, Et3N, TBAF, RT, 20h, 11%.
Scheme 1. Synthesis of FTECMO (11, (E)-3-((2-(fluoromethyl)thiazol-4yl)ethynyl)cyclohex-2-enone O-methyl oxime).
3.3
Results
3.3.1 Chemistry
The syntheses of four novel mGluR5 ligands (11, 14, 24 and 25) containing thiazole
moieties were achieved in satisfactory overall yields, however, none of the synthetic steps
was optimized. Reference 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 intermediate 1327. Compound 10 was obtained according
to the procedure previously described22. 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 coupled to the trans- isomer of 9 28 to afford
end product 11 in 11% yield. In a similar manner, 2-bromothiazole (12) was coupled to
starting material 13, which was prepared according to the procedure recently described26,
to afford 14 in moderate yield (Scheme 2).
S
S
Br
N
+
N
12
a
OCH 3
13
Reagents and conditions: (a) Pd(PPh3 )4 , CuI, Et 3N, DMF, RT, 48h, 26%.
Scheme 2. Synthesis of model compound 14.
N
N
14
OCH 3
Chapter 3
91
The Sonogashira coupling of the trans-oxime 17, that was synthesized as previously
described26, to either 2-methyl-4-bromothiazole or 4-bromothiazole gave acceptable
yields of compounds 18 and 19. Both intermediates were reacted with (2-(2bromoethoxy)ethoxy)(tert-butyl)dimethylsilane to afford the TBS protected intermediates
20 and 21. Both compounds were desilylated using TBAF and reacted with
benzylsulfonylchloride to give tosylates 22 and 23. Nucleophilic substitution reaction of
the tosyl leaving group with dry TBAF afforded final products 24 and 25.
OH
S
N
R
R
N
a
S
Br
N
15: R1=H
16: R2=CH3
N
+
17
b
OH
18: R1=H
19: R2=CH3
S
R
N
N
c
O
O
OTBS
20: R1=H
21: R2=CH3
S
R
N
N
d
O
O
OTos
22: R1=H
23: R2=CH3
S
R
N
N
O
O
F
24: R1=H
25: R2=CH3
Reagents and conditions: (a) DMF, Pd(PPh 3) 4, CuI, Et3 N, RT, 24h, 24-54%; (b) DMF, NaH, (2-(2bromoethoxy)ethoxy)(tert-butyl)dimethylsilane, RT, 2h, 72-80%; (c) 1. THF,TBAF, RT, 1.5h; 2. CH 2Cl2,
toluenesulfonylchloride, 0 °C, RT, 12h, 85-91%, (d) THF, TBAF, 60 °C, 5h, 25-28%.
Scheme 3. Syntheses of two thiazole analogues (24 and 25) of the mGluR5 radioligand 7
(Fig.1).
92
The precursor for the preparation of the radiolabeled analogue of compound 11 was
synthesized in analogy to the precursor for the synthesis of [18F]SP20322. Compound 26
was prepared according to the procedure reported by Siméon et al.22. Sonogashira
coupling of compounds 26 and intermediate 13 was accomplished in reasonable yield. A
mixture containing compound 27 was treated with TBAF to deliver alcohol 28. In a final
step the alcohol was converted to the bromide precursor 29 using CBr4 and PPh3.
I
S
N
OCH3
S
a
+
N
N
TBSO
N
OCH 3
OTBS
13
26
27
S
S
Br
b
HO
N
27
N
N
c
N
OCH 3
OCH3
28
29
Reagents and conditions: (a) DMF, Et3 N, Pd(PPh3 )4 , CuI, RT, 24h, ~ 91% (crude);
(b) THF, TBAF in THF (1M), RT, 50 min, 47%; (c) THF, CBr4 , P(Ph)3 , RT, 2h, 28%.
Scheme 4. Synthesis of the labeling precursor (E)-3-((2-(bromomethyl)thiazol-4yl)ethynyl)cyclohex-2-enone O-methyl oxime (29).
Chapter 3
93
3.3.2 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.
Ligand Stuctures of the compounds
11
14
24
25
Ki [nM]
5.5 ± 1.1
20.3 ± 6.3
36.0 ± 11.1
21.9 ± 7.0
Table 1. Binding affinity of four thiazole derivatives 11, 14, 24 and 25 towards mGluR5.
Figure 2. Displacement binding curve of FTECMO (11).
94
The fluoro-methyl-thiazole analogue, 11, showed a Ki value of 5.5 ± 1.1 nM, whereas 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 chains exhibited lower binding affinities towards mGluR5 as well but
compound 25 showed a slightly higher affinity. Since compound 11 displayed the highest
binding affinity of all four candidates tested, it was selected for radiolabeling and further
evaluation as a PET ligand for imaging mGluR5.
3.3.3 Radiosynthesis of [18F]-FTECMO
S
S
Br
a
N
18 F
N
N
N
OCH 3
OCH 3
29
[18 F]-FTECMO
Reagents and conditions: (a) K[18F]F-K2.2.2, ACN, 90 °C, 10 min.
Scheme 5. Radiosynthesis of [18F]-FTECMO.
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.
The total synthesis time was 60 min and the final product was obtained in a radiochemical
yield of up to 45% (decay corrected). Radiochemical purity was greater than 99% (Fig. 3)
and the specific activities lay between 50 and 120 GBq/µmol after quality control for
each production (n = 6). The identity of the radioligand was confirmed by coinjection of
reference 11.
3.3.4 Determination of LogDpH7.4
The lipophilicity of [18F]-FTECMO was determined by the shake flask method at
physiological pH and revealed a logDpH7.4 of 1.6 ± 0.2. This value is comparable to the
calculated logP value of 1.9.
Chapter 3
Figure 3. RadioHPLC profile of [18F]-FTECMO after semipreparative HPLC purification.
3.3.5 In vitro stability
Stability studies in rat or human plasma revealed no radioactive degradation products of
[18F]-FTECMO for up to 120 min incubation at 37°C. After 60 min of incubation in rat
liver microsomes still 93% of the parent compound remained intact. Only one radioactive
degradation product, more polar than the parent compound, was detected based on HPLC
analysis.
3.3.6 In vitro autoradiography
Incubation of rat brain slices with [18F]-FTECMO using two different 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 cerebellum was negligible.
Furthermore, blocking studies in which the rat brain slices were incubated with a mixture
of [18F]-FTECMO solution and unlabeled ABP688 led to substantially reduced and
homogeneous accumulation of activity in the rat brain slices.
95
96
Figure 4. In vitro autoradiography of horizontal sections of rat brains incubated under
baseline (A1 and B1) and blocking conditions (A2 and B2).
3.3.7 PET Study
A dynamic PET study in a Wistar rat was undertaken to analyze whether mGluR5expressing regions in the rat brain could be visualized by [18F]-FTECMO. Summed
images for the total acquisition time of 60 min did not reveal substantial brain uptake but
were characterized by high skeletal accumulation of radioactivity (data not shown). Even
in early time frames immediately after tracer injection, radioactivity accumulation in the
brain was insufficient to yield a specific signal.
Figure 5. MIP (maximum intension projection) PET images of a rat brain obtained with
[18F]-FTECMO.
Chapter 3
Figure 6. Time activity curves of [18F]-FTECMO uptake.
ROI analysis of dynamic PET data confirmed that radioactivity uptake in the skeleton
strongly increased during the duration of the scan (Fig. 5 and Fig. 6). In contrast, activity
uptake in the brain was low during the duration of the whole scan. Activity accumulation
in the cerebellum was higher than in forebrain regions (cortex, striatum, hippocampus)
suggesting non-specific uptake of radioactivity.
97
98
3.4
Discussion
MGluR5 has been shown to be involved in numerous central nervous system disorders.415
Non-invasive in vivo imaging of the receptor by using PET will give further insight into
the underlying pathophysiological processes and help to understand mGluR5
pharmacology, which in turn will speed up drug development29, 30. A lot of effort has
therefore been made in the recent years to develop suitable mGluR5 PET tracers.
However, to date only two mGluR5 PET ligands, [11C]-ABP688 and [18F]SP203, have
been fully characterized in human studies. Although [11C]-ABP688 displays favorable in
vivo imaging parameters, the labeling with the short-lived nuclide carbon-11 (t1/2 = 20
min) limits its widespread use. [18F]SP203 showed species dependent in vivo
defluorination in monkeys22 and in rats31 that is undesirable for in vivo imaging.
However, in human studies enzymatic defluorination was negligible.23, 32 In the hope of
obtaining an fluorine-18 labeled PET tracer with structural elements of both [18F]SP203
and [11C]-ABP688, novel thiazole containing ABP688 derivatives (11, 14, 24 and 25)
were synthesized and screened for their potential to bind to mGluR5. Compound 14 does
not contain a fluorine atom and served only as a model compound. Compound 11, named
FTECMO, combines structural elements from SP203 and ABP688. FTECMO exhibited a
similar binding affinity as ABP688 with a Ki value of 5.5 ± 1.4 nM compared with a 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 methyl-thiazole moiety was
obviously well tolerated by the mGluR5. However, this does not hold true for analogues
of compound 7, that was previously shown to be high affinity mGluR5 ligand. The
thiazole analogues 24 and 25 displayed decreased binding affinity for mGluR5. For these
compounds, the affinity was reduced by seven- and ten-fold, 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 PET tracer candidate. An efficient single-step radiolabeling with
fluorine-18 of the bromo-precursor 29, which was synthesized in analogy to a literature
procedure22, was successfully established. Reacting 29 with K[18F]F-K222 in acetonitrile at
90°C gave [18F]-FTECMO in a good radiochemical yield (45%, decay corrected). Semipreparative HPLC purification resulted in highly pure product with radiochemical purity
greater than 99%. A specific activity up to 120 GBq/µmol was obtained. The tracer was
identified by co-injection of unlabeled FTECMO.
Chapter 3
In vitro autoradiographical studies using rat brain slices revealed heterogeneous and
displaceable binding of [18F]-FTECMO in mGluR5-rich brain regions such as the
hippocampus, striatum and cortex. In addition, [18F]-FTECMO exhibited high stability in
human and in rat plasma over 120 min. In rat liver microsomes, only low degradation was
observed. The short retention time in the UPLC system points to a very hydrophilic
degradation product. The radioactivity detected is presumably derived from [18F]fluoride. Similar observations were made with 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP), a structurally related mGluR5 antagonist, when it was incubated with
mouse liver microsomes.33
In vivo PET imaging of a Wistar rat injected with [18F]-FTECMO showed minimal
radioactive uptake into the brain. Accumulation of activity in the bones, including the
skull, was observed and increased during the scan. This is a strong evidence for in vivo
radiodefluorination34. Visualization of mGluR5-rich brain regions in vivo was not
possible because radioactive uptake in the brain was not sufficient for a clear PET signal
with an appropriate signal-to-background ratio. Even for early time points immediately
after tracer injection, the accumulation of radioactivity in the brain was insufficient to
yield a clear signal (Fig. 5). However, the lipophilicity of [18F]-FTECMO (logDpH7.4 = 1.6
± 0.2) determined by the shake-flask method suggests ideal physicochemical properties
for diffusion through the blood-brain barrier35. Therefore the low brain uptake may be
related to the rapid radiodefluorination of [18F]-FTECMO. ROI analysis of dynamic PET
data confirmed uptake into the bones and demonstrated that activity in the forebrain
(cortex, striatum, hippocampus) and the hindbrain (cerebellum) peaked negligibly
immediately after radiotracer injection. The low levels of activity in the brain reveal no
specific binding of [18F]-FTECMO to mGluR5 rich brain regions. Only recently, PET
imaging with [18F]-FPECMO, a fluoropyridine analogue of ABP688, similarly showed no
proper visualization of mGluR5 rich brain regions in rats. [18F]-FPECMO was rapidly
defluorinated in vivo and resulted in radioactivity accumulation in bones and the skull24.
The radioactivity uptake into rat brain after [18F]-FPECMO injection peaked early after
tracer injection but was eliminated from the fore- and hindbrain regions quickly. In the
[18F]-FTECMO PET scan, this initial peak was not observed. With regard to the terms of
the kinetic profile and the metabolic clearance of [18F]-FTECMO, it is noteworthy that
[18F]-FE-DABP688, the first fluorine-18 labeled ABP688 analogue, was also rapidly
washed out from the brain of rats.25 The retention of the tracer in mGluR5 rich brain
regions, however, was more favorable compared with [18F]-FTECMO.
99
100
In terms of both the methylthiazole moiety as structural element and the labeling position,
[18F]-FTECMO is more comparable to [18F]SP203. Enzymatic degradation of [18F]SP203
was reported to occur in the rat brain and in the periphery. The mechanism of
radiodefluorination was shown to be mediated by glutathionylation through the
glutathione transferase system31. Radiodefluorination in [18F]-FTECMO is most likely
derived from similar metabolic interactions. The structural similarity of the [18F]fluorothiazole moiety in both tracers points to a similar metabolic profile. However, in
2008 Brown et al.23 described the successful in vivo imaging in humans with [18F]SP203,
which was not hindered by in vivo defluorination. Glutathione transferase was recently
identified as the responsible enzyme for this radiodefluorination31. This enzyme is known
to dehalogenate alkyl chlorides, bromides and iodides36. Presumably, mGluR5 PET
imaging in humans using [18F]SP203 and the resulting proper visualization of the receptor
was due to species and tissue differences of the mammalian glutathione transferases. In
analogy to the metabolic profile of [18F]SP203 the radiodefluorination of the [18F]fluoromethythiazole moiety of [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.
3.5 Conclusion
In summary, the syntheses and investigation of four novel thiazole containing ABP688
analogues led to the identification of a high binding affinity ligand for mGluR5,
FTECMO (11). 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.
Chapter 3
3.6
Experimental
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 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
spectra: singlet (s), doublets (d), triplet (t), quartet (q), quintet (quint), doublet of doublets
(dd), mulitplet (m). The chemical shifts of complex multiplets are given as the range of
their occurance. Low-resolution 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
reversed phase column (Gemini 10 µm C18, 300 x 3.9 mm, Phenomenex) by applying an
isocratic solvent system with 70% MeCN in water and a flow of 1 mL/min. In vitro
stability tests were performed on a Waters Acquitiy UPLC system (Berthold
radiodetector) occupying an Acquitiy UPLC BEH C18 1.7 µm (2.1 x 50 mm) column. 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 3 minutes a gradient (100 % B
to 100% A) was applied followed by 0.5 minutes under isocratic conditions (100% A).
The Swiss Federal Veterinary Office approved animal care and all experimental
procedures.
3-bromocyclohex-2-enone O-methyl oxime (9)
O-methylhydroxylamine hydrochloride (0.8 g, 9.4 mmol) was added to a solution of
crude 9 (1.09 g, 6.27 mmol) in pyridine (20 mL). After stirring at RT for 18 h, 30 mL of
H2O were added and the reaction mixture was extracted with ether. The combined organic
layers were washed with saturated CuSO4 solution (3 x 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
101
102
mg, 63%). trans isomer : 1H-NMR (400 MHz, CDCl3): δ 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). cis isomer : 1HNMR (400 MHz, CDCl3): δ 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).
(E)-3-((2-(fluoromethyl)thiazol-4-yl)ethynyl)cyclohex-2-enone O-methyl oxime (11).
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-(fluoromethyl)-4-((trimethylsilyl)ethynyl)thiazole (85 mg, 0.6 mmol) in DMF (4
mL). After stirring for 30 min, the mixtures were combined and stirred for 20 h at
RT. Sat. NH4Cl solution (20 mL) was added and the resulting mixture was extracted with
EtOAc (3 x 20 mL). The combined organic layers were washed with water (2 x 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%).
1
H NMR (400 MHz, CDCl3): δ 7.53 (s, 1H), 6.54 (s, 1H), 5.67 (s, 1H), 5.58 (s, 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): δ 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): δ -211.52. MS m/z 264.94 (M + H)+. HRMS calcd for C13H13FN2OS,
264.0728; found, 264.0728.
(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 degassed solution of 2bromothiazole 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 described26. The mixture was stirred at room temperature for 48 h, quenched
with sat. NH4Cl (20 mL) and extracted with ether (3 x 20 mL). The combined organic
layers were washed with water (2 x 20 mL) and brine (20 mL). The solvents were
evaporated under reduced pressure and the residue was purified by column
Chapter 3
103
chromatography with pentane/EtOAc (9:1) to afford compound 14 as light yellow crystals
(74 mg, 26%).
1
H NMR (400 MHz, CDCl3): δ 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): δ 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 C12H13N2OS,
233.0743; found, 233.0744.
(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 chromatography using
pentane/EtOAc (1:1) as the mobile phase.
1
H NMR (400 MHz, CDCl3): δ 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).
13
C NMR (100 MHz,
CDCl3): δ 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)+.
(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%).
1
H NMR (400 MHz, CDCl3): δ 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).
13
C NMR (100 MHz,
CDCl3): δ 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, 232.06703; found, 232.0665.
(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)(tertbutyl)dimethylsilane (340 mg, 1.2 mmol) was added. The reaction mixture was stirred for
1.5 h and then quenched by addition of a 50 % NaHCO3 solution (15 mL). The mixture
104
was extracted with ether (3 x 20 mL). The combined organic layers were washed with
water (2 x 20 mL) and brine (20 mL) and dried over Na2SO4 . The solvents were
evaporated 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%).
1
H NMR (400 MHz, CDCl3): δ 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): δ -5.3, 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.
(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 obtained as a clear oil (315 mg, 72%).
1
H NMR (400 MHz, CDCl3): δ 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): δ -5.3, 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)+.
(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 under 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 Et3N (312 µl). The mixture was
cooled to 0°C and then treated with toluenesulfonylchloride (401 mg, 1.3 mmol). After
Chapter 3
105
stirring at RT for 8 hours, the mixture was diluted with water (25 mL) and extracted with
ether (3 x 20 mL). The combined organic layer was washed with water (2 x 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 pentane/Et2O (1:4) to
give pure 22 as a yellow oil ( 360 mg, 91%).
1
H NMR (400 MHz, CDCl3): δ 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).
13
C NMR
(100 MHz, CDCl3): δ 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.
(E)-2-(2-(3-((2-methylthiazol-4-yl)ethynyl)cyclohex-2enylideneaminooxy)ethoxy)ethyl 4-methylbenzenesulfonate (23).
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%).
1
H NMR (400 MHz, CDCl3): δ 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).
13
C NMR (100 MHz, CDCl3): δ 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, 475.13559; found, 475.1356.
(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 vacuum at 45°C for 24 h and
was then dissolved in dry THF (10 mL). A solution of 22 (180 mg, 0.4 mmol) in dry 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 x 20 mL). The organic layers were washed with
water (2 x 20 mL) and brine (20 mL) and dried over Na2SO4. After evaporation of the
solvents, the brown crude product was purified by column chromatography with
Et2O/pentane (6:1) to give compound 24 as a clear oil (26 mg, 21%).
1
H NMR (400 MHz, CDCl3): δ 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 =
106
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).
13
C NMR (100 MHz, CDCl3): δ 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.
19
F NMR (376
MHz, CDCl3): δ -223.09. MS m/z 308.80 (M + H)+.
(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. Starting material 23 (150 mg,
0.32 mmol) gave a slightly yellow oil (39 mg, 40%).
1
H NMR (400 MHz, CDCl3): δ 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): δ 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.
19
F
NMR (376 MHz, CDCl3): δ -223.11. MS m/z 322.77 (M + H)+. HRMS calcd for
C16H19N2O2SFNa+, 345.10435; found, 345.1044.
(E)-3-((2-((tert-butyldimethylsilyloxy)methyl)thiazol-4-yl)ethynyl)cyclohex-2-enone
O-methyl oxime (27).
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%).
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.
(E)-3-((2-(hydroxymethyl)thiazol-4-yl)ethynyl)cyclohex-2-enone
O-methyl
oxime
(28).
To a solution of 27 containing impurities (920 mg, 2.443 mmol) in THF (25.5 mL) was
added a solution of TBAF (1M) 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
extracted with EtOAc (3 x 20 mL) and the combined organic layers were washed with
water (2 x 20 mL) and brine (20 mL) and dried with Na2SO4. The solvents were
evaporated under reduced pressure and the residue was purified by flash column
Chapter 3
107
chromatography with pentane/EtOAc (6:4) to give the desired alcohol 28 (304 mg, yield
= 47 %). 1H NMR (400 MHz, CDCl3): δ 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): δ 20.8, 22.1, 29.4, 62.0, 62.1, 86.5, 90.0, 123.0,
127.2, 130.3, 137.1, 155.4,171.5. MS m/z 263.02 (M + H)+. HRMS calcld for [M+]
C13H14FN2O2S+, 262.0771; found: 262.0768.
(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 mixture 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 solid (89 mg, 30%). 1H NMR (400 MHz, CDCl3): δ 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).
13
C NMR (100 MHz, CDCl3): δ 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 calcld for
[M+] C13H13BrN2OS+, 323.9927; found: 323.9930.
Preparation of membranes. Sprague Dawley rats were sacrificed by decapitation and
the brains were harvested immediately. After separation of the cerebellum 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 (1000 g, 15 min, 4 ˚C) to give a
pellet (P1). The supernatant was removed and kept while P1 was resuspended in 5
volumes of sucrose buffer. After homogenization and centrifugation of the suspension the
new supernatant was collected and combined with the previous supernatant.
Centrifugation of the obtained mixture (17000 g, 20 min, 4 ˚C) resulted in pellet (P2),
which was resuspended with incubation buffer (5 mM Tris/acetate buffer, pH 7.4). This
suspension was again centrifuged (17000 g, 20 min, 4 ˚C) followed by resuspension of
the resulting pellet with incubation buffer to obtain the final membrane preparation
suitable for storage at -70 ˚C. For each assay the required amount of membrane
preparation was thawed and kept on ice during all preparation processes. In order to
108
determine the protein concentration, a Bio-Rad Microassay with bovine serum albumin as
a standard protein (Bradford, 1976)37 was accomplished before each experiment.
Displacement assays. For all four novel ligands (11, 14, 24 and 25) the binding affinity
was determined by displacement assays using [3H]-M-MPEP (2 nM) as a radioligand.
The P2 membranes were incubated with increasing concentrations of the test compound
(1 pM – 100 µM), each in triplicate, in incubation buffer II (30 mM NaHEPES, 110 mM
NaCl, 5 mM KCl, 2.5 mM CaCl2xH20, 1.2 mM MgCl2, pH 8). Non-specific binding was
determined in the presence of ABP688 (100 µM). The test samples with a total volume of
200 µL were incubated for 45 min at RT. For the separation of free radioligand, 4 mL of
ice-cold incubation buffer II were 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 and transferred
into beta scintillation vials. Radioactivity retained on the filters was measured by beta
counting (Liquid Scintillation Analyser 1900TR, Canberra Packard). The set of data was
evaluated with KELL Radlig Software (Biosoft). For calculation of the Ki value, the
Cheng-Prusoff equation was applied.
Radiosynthesis of [18F]-FTECMO. The conversion of 30 into [18F]-FTECMO was
achieved via nucleophilic substitution with [18F]-fluoride. [18F]-fluoride was obtained via
the 18O(p,n)18F reaction using 98% enriched 18O-water. 18F- 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 K222 solution
(Kryptofix K222; 2,5 mg, K2CO3, 0,5 mg in MeCN/water (3:1)) was used for the elution of
18 -
F from the cartridge. The solvents were evaporated at 110 ºC under vacuum in the
presence of slight inflow of nitrogen gas. After addition of acetonitrile (1 mL), azeotropic
drying was carried out. This procedure was repeated twice and gave rise to dry K222K[18F] F complex. A solution of precursor 30 (2 mg in 300 µl of dry acetonitrile) was
added to the dried K222-K[18F] F complex . The reaction mixture was heated at 90ºC for
10 min, followed by the addition of 50% MeCN in water (2 mL) . Purification by
semipreparative HPLC was carried out on a HPLC system equipped with a Merck-Hitachi
L-6200A intelligent pump, a Knauer Variable Wavelength Monitor UV detector and a
Geiger Müller LND 714 counter with Eberlein RM-14 instrument using a reversed phase
column (Gemini 5µ C18, 250 x 10 mm, Phenomenex) with a solvent system and gradient
Chapter 3
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. Ascorbic acid (16 mg) was added to the
fraction containing [18F]-FTECMO, and the mixture was diluted 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. In
order to obtain an injectable solution, the fraction was diluted with saline water (9.5 mL)
and sterile filtration was performed. Determination of relative lipophilicity, radiochemical
purity and specific activity was carried out during the quality control step. Identification
of [18F]-FTECMO was achieved by co-injection of reference 11.
Determination of LogDpH7.4. The shake flask method38 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 of 20 µL of [18F]-FTECMO solution (80
kBq) the samples were mixed for 15 min and then centrifuged at 5000 rpm for 5 min
before the radioactivity in each phase was determined using a gamma counter (Wizard,
Perkin Elmer).
In vitro stability tests. For the determination of the in vitro stability of the radioligand,
13.5 µL of [18F]-FTECMO in ethanol were added to human and rodent plasma (386.5
µL). The solutions were incubated over 120 minutes at 37 ˚C. At five different time
points (0, 30, 60, 90, and 120 minutes) aliquots (70 µL) were taken and transferred into
ice-cold acetonitrile (140 µL). After 10 min of centrifugation (13000 rpm, 4 ˚C), the
supernatant was analyzed by UPLC applying the conditions described above.
In addition, [18F]-FTECMO stability was investigated employing pooled rat liver
microsomes (BD Bioscience). The test compound (20 – 50 nM) or 15 µM testosterone
(positive control), respectively, were preincubated 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 60 min) 150 µL of ice-cold acetonitrile were added in
order to precipitate all active enzymes. The samples were centrifuged at 13000 rpm for 5
min to obtain the supernatant to be analyzed by UPLC as described.
109
110
In vitro autoradiography. The experiments were carried out with rat brain slices of male
Sprague Dawley rats. The rats were sacrificed by decapitation and the brains were
removed quickly and frozen in 2-methylbutane (Fluka) at -30 to -36˚C. Horizontal brain
slices (10 µm) were obtained by cutting the brains at -20˚C with a Cryostate microtome
HM 505N (Microm). The slices were absorbed on SuperFrost slides (Menzel) and stored
at -80˚C until used. For the experiment, the slices were allowed to thaw at RT for 30 min
before incubation in HEPES-BSA buffer (30 mM Na-HEPES, 110 mM NaCl, 5 mM KCl,
2.5 mM CaCl2 x H2O, 1.2 mM MgCl2, 0.1% BSA, pH 7.4) at 0˚C for 10 min. The brain
slice was then dripped with 300 µL of a [18F]-FTECMO solution (0.5 nM or 5 nM,
respectively) and incubated for 45 min at RT in a humid chamber. For blockade
conditions, the brain slices were dripped with 300 µL of a mixture of unlabeled ABP688
and [18F]-FTECMO (10 µM ABP688, 0.5 nM or 5 nM, respectively, [18F]-FTECMO) and
incubated in the same way for 45 min. After decanting the supernatant tracer solution, the
brain slices were washed with HEPES buffer for 3 min (3 x) and with distilled water for 5
seconds (2 x ) at 0 ˚C. A drying step at RT (40 min) was followed by exposition (7 min)
of the brain tissue to appropriate phosphor imager plates (AGFA) and scanning in a
BAS5000 reader (Fuji).
In vivo PET imaging. Animal care and all experimental procedures were approved by the
Cantonal Veterinary Office. An adult male Wistar rat (436 g) was obtained from Charles
River (Sulzfeld, Germany) and was allowed free access to food and water. PET scanning
was performed using the GE VISTA PET/CT tomograph, which is characterized by high
sensitivity but a limited axial field-of-view of 4.8 cm39. The animal was immobilized by
an isoflurane inhalation anesthesia and fixed on the bed of the tomograph before tracer
injection. Monitoring of anesthesia during scanning was performed according to protocols
published previously40. The animal was injected intravenously with [18F]-FTECMO (30
MBq, <0.138 nmol) and scanned in a single bed position (setting the brain in the center of
the field-of-view) with a dynamic PET acquisition mode for 60 min. Raw data were
acquired in list-mode and reconstructed in user-defined time frames (dynamic: 5x2, 6x5,
2x10 min; static: 1x60 min) with a voxel size of 0.3775x0.3775x0.775 mm and a matrix
size of 175x175x61. Image files were evaluated by region-of-interest (ROI) analysis
using the dedicated software PMOD41.
Chapter 3
Acknowledgment. We acknowledge the technical support of Claudia Keller, Mathias
Nobst, Lukas Dialer. We thank Christophe Lucatelli, Stefanie-Dorothea Krämer and
Harriet Struthers for fruitful discussion.
3.7
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115
116
4. In vitro and in vivo Evaluation of
[18F]-FDEGPECO as Novel
Fluorine-18 Labeled PET Tracer for
Imaging Metabotropic Glutamate
Receptors Subtype 5
118
Chapter 4
119
4.1
Abstract
[18F]-FDEGPECO,
(E)-3-(pyridin-2-ylethynyl)cyclohex-2-enone
O-2-(2-18F-
fluoroethoxy)ethyl oxime, a novel high affinity radioligand for the metabotropic
glutamate receptor of the subtype 5 (mGluR5) was assessed for its potential as a PET
imaging agent. In vitro characterization of [18F]-FDEGPECO included the confirmation
of stability in human and in rodent plasma as well as in human liver microsomes. Using a
cell layer of human P-gp transfected hMDR1-MDCKII cells (Madin-Darby canine kidney
cells), [18F]-FDEGPECO was shown not to be a P-gp substrate. The heterogeneous and
displaceable uptake of radioactivity observed in mGluR5-rich brain regions such as
hippocampus and striatum from the in vitro autoradiographical analyses of [18F]FDEGPECO on rat brain slices encouraged the in vivo evaluation of the tracer. PET
imaging in Wistar rats revealed the same radioactivity uptake pattern in the rat brain with
highest accumulation in the hippocampus. The lowest uptake was observed in the
cerebellum, a brain region with negligible mGluR5 expression. In vivo blockade PET
studies with M-MPEP (1mg/kg) resulted in a reduced and homogeneous uptake of
radioactivity in the rat brain, indicating specificity of [18F]-FDEGPECO binding to
mGluR5. Postmortem biodistribution studies carried out at 15 min post injection
confirmed the distribution pattern observed in the PET images obtained with [18F]FDEGPECO. Uptake of radioactivity in mGluR5-rich brain regions such as hippocampus,
striatum and cortex were 0.18, 0.16 and 0.13%IDnorm./g, respectively. The
hippocampus-to-cerebellum and the striatum-to-cerebellum ratios were 2.57 and 2.89,
respectively. Co-injection of [18F]-FDEGPECO with M-MPEP (1 mg/kg) revealed a 50%
reduced binding of [18F]-FDEGPECO in mGluR5-rich brain regions. It is anticipated that
the further evaluation of [18F]-FDEGPECO in humans might yield PET images of even
higher quality due to the stronger and longer-lived signals.
120
4.2
Introduction
Glutamate receptors encompass a large and heterogeneous family of ionotropic as well as
metabotropic glutamate receptors (mGluRs). The discovery of the latter started with the
identification of mGluR1 in 19911. To date, eight different receptor subtypes have been
cloned. They are classified into three groups according to their sequence homology,
secondary messenger system and receptor pharmacology. The mGluR1 and mGluR5 form
group I. MGluR2 and mGluR3 belong to group II and mGluR4, 6, 7 and 8 are members
of group III2-4. The mGluR5 has been suggested to be involved in numerous central
nervous system (CNS) disorders such as anxiety, schizophrenia, depression, neuropathic
pain, drug addiction and Parkinson`s disease5-11. Therefore, mGluR5 is of high interest as
a potential drug target for the treatment of these diseased conditions. The non-invasive
positron emission tomography (PET) imaging of CNS receptors and of changes in
receptor density is known to accelerate drug development12. So far a number of PET
tracers for imaging mGluR5 have been reported. To date, [11C]-ABP688 (2, Fig 1) and
[18F]SP203 (4, Fig.1) are the most successful and best characterized PET tracers for
imaging mGluR5 in humans.
N
N
O
O
18
F
1 , [18 F]-FDEGPECO
H 3C
N
N
O 11 CH 3
2 , [11 C]-ABP688
S
N
N
O
18F
F
N
F
N
4 , [18 F]SP203
3, [18 F]-FE-DABP688
18
18
F
CN
N
N
18 F
OCH 3
5 , [18 F]-FPECMO
Figure 1. Structures of mGluR5 PET tracers.
6 , [18 F]F-PEB
CN
Chapter 4
121
Very recently, the use of [18F]F-PEB (6, Fig 1) in human subjects was also reported13.
Although these tracers are useful, they are plagued with some shortcomings. [18F]F-PEB
was obtained in low radiochemical yields and [18F]SP203 (4, Fig.1) displayed high
radiodefluorinaton in rats and monkeys14,
15
, although negligible defluorination was
observed in human subjects15. [11C]-ABP688 (2, Fig 1), the first successful PET tracer for
imaging mGluR5 in humans, is one of the most interesting mGluR5 PET tracers due to its
pharmacokinetic profile. However, its widespread use is limited due to the short physical
half life of carbon-11 (t1/2 = 20 min). Our group recently reported on the evaluation of
[18F]-FE-DABP688 (3, Fig. 1) and [18F]-FPECMO (5, Fig.1)16,
17
, both fluorinated
derivatives of ABP688. Although both compounds showed excellent in vitro properties,
their in vivo characteristics were not optimal. More recently, we have identified a new
ABP688 derivative herein called [18F]-FDEGPECO (1, Fig. 1). This compound displays
high in vitro affinity for mGluR5 (Ki = 3.8 ± 0.4 nM) and optimal lipophilicity (logDpH7.4
= 1.7 ± 0.1) for penetration through the blood-brain barrier18. Herein, we report on the in
vitro and the in vivo evaluation of [18F]-FDEGPECO as a potential PET tracer for
imaging mGluR5.
4.3
Materials and methods
General. All chemicals unless otherwise stated were purchased from Sigma-Aldrich or
Merck and used without further purification. High-performance liquid chromatography
(HPLC) analyses were performed using a reversed phase column (Gemini 10 µm C18,
300 x 3.9 mm, Phenomenex) with an isocratic solvent system 70% MeCN in water, flow
rate: 1 mL/min. Analytical HPLC chromatograms were obtained using an Agilent 1100
systems with Gina software, equipped with UV multi-wavelength and Raytest Gabi Star
detectors. Semi-preparative HPLC purifications were carried out using a reversed phase
column (Gemini 5 µm C18, 250 x 10 mm, Phenomenex) under the following conditions:
H2O (solvent A), MeCN (solvent B); 0-3 min, B 5%; 3-6 min B 5% → B 50%; 6-20 min
B 50%, flow rate: 5 mL/min. Semi-preparative HPLC system used was a Merck-Hitachi
L6200A system equipped with Knauer variable wavelength detector and Eberline
radiation detector. For the in vitro stability studies, an Ultra-performance liquid
chromatography (UPLC™) system from Waters with a Waters Acquity UPLC BEH C18
122
column (2.1 x 50 mm, 1.7 µm) and an attached Berthold co-incidence detector (FlowStar
LB513) was used.
4.3.1 Radiosynthesis of [18F]-FDEGPECO
The synthesis of [18F]-FDEGPECO has been described in detail elsewhere18. Briefly, the
corresponding tosyl-precursor was reacted with dried K222-K[18F]F complex in DMF at
90°C for 10 min. Purification by semi-preparative HPLC resulted in a solution of [18F]FDEGPECO in acetonitrile/water (1:1). After dilution with 15 mL of water, the solution
was passed over a C18 light SepPak cartridge, which was preconditioned with 5 mL of
ethanol followed by 5 mL of water, for trapping [18F]-FDEGPECO. The cartridge was
washed with 5 mL of water in order to remove traces of MeCN and the product was
eluted with 0.5 ml of ethanol. After evaporation of the solvent, the product was
formulated for intravenous application.
4.3.2 In vitro stability test
For the determination of the in vitro stability of the radioligand, 13.5 µL of [18F]FDEGPECO solution were added to human and rodent plasma (386.5 µL). The mixture
was vortexed and five aliquots of 70 µL were taken and incubated at 37˚C in Eppendorf
cups. At five different time points (0, 30, 60, 90 and 120 min) ice cold acetonitrile (140
µL) was added to one of the five samples in order to inactivate and precipitate the
proteins. After 10 min of centrifugation (13000 rpm, 4˚C), the supernatant of each sample
was analyzed by analytical HPLC.
In addition, the stability of [18F]-FDEGPECO was investigated employing pooled human
liver microsomes (BD Bioscience). The test compound (20 – 50 nM) or 15 µM
testosterone (positive control), respectively, were pre-incubated for 5 min with 1 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 for negative
control. At four different time points (0, 15, 30 and 60 min) 150 µL of ice-cold
acetonitrile were added. The samples were centrifuged at 13000 rpm for 5 min to obtain
the supernatant for UPLC analysis.
Chapter 4
4.3.3 P-glycoprotein (P-gp) substrate test
To evaluate passive permeation across a tight cell layer and to investigate whether [18F]FDEGPECO is a transport substrate of P-gp, its permeation across human P-gp
transfected hMDR1-MDCKII cells19, kindly provided by the Netherlands Cancer
Institute, was investigated. The cells were cultured at 37°C in Dulbecco`s modified
Eagle`s medium with Glutamax-I (DMEM, Gibco-BRL) supplemented with 50 units
penicillin/ml and 50 µg streptomycin/ml (penicillin-streptomycin, #15140 Gibco-BRL)
and 10% fetal calf serum (FCS, Gibco-BRL). For transport experiments, 500`000
cells/cm2 were seeded on polyethylene terephthalate culture inserts (Falcon #35-3090, 4.2
cm2, 0.4 µm pores) in 6-well plates three days prior to the experiment. The culture
medium was added to both the apical (2.5 mL) and basal (3.0 mL) compartments and was
renewed on days one and two. On day three, the medium was replaced on both sides with
pre-warmed DMEM containing 1% FCS or by pre-warmed DMEM / 1% FCS containing
40 μM P-gp modulator verapamil (added as a 2 mM stock solution in 4% ethanol). [18F]FDEGPECO (400 kBq, ca 1 nM) was added to the apical or basal compartments of three
inserts each without verapamil and three inserts each containing verapamil. The plates
were incubated at 37°C on a rocking plate and 150 μL samples were withdrawn from the
receiver compartments 20, 40, 60, and 120 min after addition of [18F]-FDEGPECO and
from the donor compartment at 120 min. As a positive control, the transport of the P-gp
substrate [3H]-vinblastine (20 kBq, 8 μM) was determined in parallel. The radioactivities
were quantified by liquid scintillation counting (LCS, Beckmann Instruments LS 6500)
and gamma counting (Wizard, Perkin Elmer), respectively. The apparent permeation
coefficient (Papp) was calculated according to the following equation (Equation 1)
Papp = ΔQr/(Δt × A) × 1/C0
where Qr is the amount [mol] of drug in the receiver compartment, t is the time [s], C0 is
the initial drug concentration [mol × cm-3] in the donor compartment and A is the area of
the cell layer (4.2 cm2). A ratio of Papp (basal to apical) to Papp (apical to basal) > 1 in the
absence of verapamil indicates P-gp transport20.
123
124
4.3.4 In vitro autoradiography
Male Sprague Dawley rats were sacrificed by decapitation and their brains were quickly
removed and frozen in 2-methylbutane (Fluka) at -30 to -36˚C. Horizontal brain slices (20
µm) were obtained by cutting the brains at -20˚C with a Cryostat microtome HM 505N
(Microm). The slices were absorbed on SuperFrost slides (Menzel) and stored at -80˚C.
For the experiment the slices were thawed at room temperature for 30 min before
incubation in HEPES-BSA buffer (30 mM Na-HEPES, 110 mM NaCl, 5 mM KCl, 2.5
mM CaCl2 x H2O, 1.2 mM MgCl2, 0.1% BSA, pH 7.4) at 0˚C for 10 min. The brain slice
was then dripped with 300 µL of a [18F]-FDEGPECO solution (0.3 nM) and incubated for
45 min at room temperature in a humid chamber. For blockade conditions, the brain slices
were incubated with 300 µL of [18F]-FDEGPECO and 10 µM ABP688. After decanting
the supernatant tracer solution, the brain slices were washed thrice with HEPES buffer for
3 min and twice with distilled water for 5 seconds at 0˚C. The slides were dried at room
temperature (40 min) and exposed for 15 min to phosphor imager plates (BAS-MS 2025,
Fuji Photo Film Co. Ltd.) which were subsequently scanned by a BAS5000 reader (Fuji).
4.3.5 In vivo characterization.
Animals (male Wistar rats) were obtained from Charles River (Sulzfeld, Germany).
Animal care and all experimental procedures were approved by the Cantonal Veterinary
Office. The animals were allowed free access to water and food.
PET studies. PET scanning was performed using the GE VISTA PET/CT tomograph.
This device is characterized by high sensitivity and an axial field of view of 4.8 cm21.
Isoflurane inhalation anesthesia was used for immobilization of the animal that was then
fixed on the bed of the tomograph before tracer injection. Monitoring of anesthesia during
scanning was performed as previously described22. One animal was injected
intravenously with [18F]-FDEGPECO (15.7 MBq, 0.08 nmol) and scanned in a wholebody configuration using four bed positions in a dynamic acquisition mode for 68 min.
The specificity of [18F]-FDEGPECO uptake in the brain was analyzed under baseline and
blockade conditions in another PET experiment using one bed position. After intravenous
injection of [18F]-FDEGPECO (26.9 MBq, 0.23 nmol) PET data were acquired under
baseline conditions in a dynamic mode for 30 min. A second Wistar rat was co-injected
with 20.5 MBq (0.26 nmol) of [18F]-FDEGPECO and M-MPEP (1 mg/kg) and scanned
accordingly. PET data were reconstructed in user-defined time frames with a voxel size of
Chapter 4
0.3775x0.3775x0.775 mm. Image files were evaluated by region-of-interest (ROI)
analysis using the dedicated software PMOD23. Time-activity curves were normalized to
the injected dose per gram of body weight and expressed as standardized uptake values
(SUV).
Postmortem biodistribution studies. [18F]-FDEGPECO was administered intravenously to
restrained Wistar rats (183-191 g) by tail vein injection. For the blockade experiments,
animals were co-injected with M-MPEP (1.0 mg/kg body weight, 1.0 mg/ml
PEG200/water = 1:1) and 200 µL of [18F]-FDEGPECO (13.9 – 23.5 MBq, 0.12 nmol).
All control animals (n = 3) were injected with 200 µL of tracer solution (14.9 – 21.9
MBq, 0.12 nmol) and a corresponding volume of vehicle. The rats were sacrificed by
decapitation 15 min post injection. The whole brain was removed and the selected brain
regions (striatum, hippocampus, cortex, cerebellum, midbrain and the brain stem) were
dissected. Blood, urine, lung, liver, kidneys, muscle, heart, bone, and spleen were taken as
well. The individual organs and the separated brain regions were weighed and the
radioactivity in each sample was measured in a gamma counter (Wizard, Perkin Elmer).
Activity concentrations were calculated as percentage injected dose normalized to body
weight per tissue weight (%IDnorm./g wet tissue).
4.4
Results
4.4.1 Radiosynthesis of [18F]-FDEGPECO
[18F]-FDEGPECO was obtained from a single-step reaction sequence as described
previously18. The total synthesis time including cartridge and semi preparative HPLC
purifications was around 45 min. Radiochemical purity was greater than 98% and the
specific activity was between 110 and 508 GBq/µmol after formulation. The
radiochemical yield (decay corrected) was 40%. The identity of [18F]-FDEGPECO was
either confirmed by co-injection of FDEGPECO or by comparison of the retention time
of non-radioactive reference molecule under the same elution conditions.
4.4.2 In vitro stability of [18F]-FDEGPECO
Stability tests were carried out in rodent and human plasma over a period of 120 min at
36°C. No radioactive degradation products of [18F]-FDEGPECO were detected.
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Microsomal samples analyzed by UPLC exhibited no additional radioactive degradation
products. After 60 min, 99% of parent compound was detected. Later time points were
not examined.
4.4.3 P-gp substrate test
P-gp is an efflux transporter present in the luminal (apical) plasma membrane of brain
capillary endothelial cells where it impedes the blood-brain barrier passage of many
pharmacologically active compounds24. To investigate whether [18F]-FDEGPECO is a
substrate for P-gp, its permeation across P-gp transfected MDCK cells was compared to
the permeation of vinblastine, a known P-gp substrate with a higher basal to apical than
apical to basal permeation25. The results are shown in Figure 2.
18
0.16
A
16
4
B
C
14
0.08
0.06
3
12
6
6
0.10
Papp [cm/s x 10 ]
0.12
Papp [cm/s x 10 ]
Fraction permeated
0.14
10
8
6
0.04
4
0.02
2
1
0
0.00
0
20
40
60
80
100
120
2
0
ab
ba
ab inh
ba inh
vin ab
vin ba
Time [min]
Figure 2. Permeation of [18F]-FDEGPECO and vinblastine across hMDR1-MDCK(II) cells.
A) [18F]-FDEGPECO (black and open symbols) or vinblastine (grey symbols) were added to
the apical (squares) or basal (triangles) compartment. [18F]-FDEGPECO permeation was
determined in the absence (open symbols) and presence (closed symbols) of verapamil. B)
ab, [18F]-FDEGPECO Papp from the apical to the basal compartment; ba, from the basal to the
apical compartment; inh, in the presence of 40 μM verapamil. C) Papp values of vinblastine
from apical to basal (ab) and from basal to apical (ba).
In contrast to vinblastine, [18F]-FDEGPECO showed higher permeation in the apical to
basal direction than from basal to apical. No significant difference was observed in the
presence of the P-gp modulator verapamil. Permeation of [18F]-FDEGPECO in both
directions was higher than vinblastine permeation. Based on these data, we conclude that
[18F]-FDEGPECO is not a transport substrate of P-gp. The relatively high Papp in apical to
basal direction, i.e. (1.3 ± 0.3) × 10-5 cm/s, points to good barrier passage.
Chapter 4
4.4.4 In vitro autoradiography
[18F]-FDEGPECO was investigated for its distribution in the rat brain in vitro. Horizontal
rat brain slices incubated with the tracer solution (0.3 nM) exhibited heterogeneous
binding with highest accumulation of radioactivity in the hippocampus, striatum and
cingulate cortex, regions known to express mGluR5 in high levels (Fig. 3A). On the
contrary, radioactivity uptake in the cerebellum, a region with negligible mGluR5 density,
was clearly lower (Fig. 3A). Rat brain slices that were co-incubated with [18F]FDEGPECO and 10 µM ABP688, a high affinity ligand for mGluR5, displayed
substantially reduced and homogeneous accumulation of radioactivity (Fig. 3B).
Figure 3. In vitro autoradiography of horizontal rat brain slices (20 μm) incubated with [18F]FDEGPECO (0.3 nM) under baseline conditions (A) and blocking conditions (B).
4.4.5 In vivo characterization
In vivo PET imaging. An anesthetized rat injected with [18F]-FDEGPECO was subjected
to a whole body PET scan. The obtained PET images exhibited highest activity
concentrations in the liver and intestines (Fig. 4) indicating that [18F]-FDEGPECO is
eliminated mainly via the hepatobiliary system.
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128
Figure 4. Series of horizontal (ventral to dorsal) whole-body PET images of a Wistar rat
injected with 15.8 (0.08 nmol) MBq of [18F]-FDEGPECO. Raw data were reconstructed and
summed from 0 to 68 min post injection.
No uptake of radioactivity was observed in the bone. Tracer uptake into the brain was
lower than uptake into excretory organs. Nevertheless, the visualization of hippocampus,
striatum and cortex was possible. As expected, the cerebellum showed negligible uptake
of radioactivity.
Figure 5. Time-activity curves of [18F]-FDEGPECO uptake in the cortex, striatum,
hippocampus (cx/st/hi) and the cerebellum (ce).
The resulting time-activity curves of the combined activity accumulation in cortex,
striatum, hippocampus and the cerebellum are presented in Figure 5. For all the brain
regions, radioactivity peaked at five min post injection. During the whole duration of the
scan, the level of activity concentration was higher in mGluR5-rich forebrain regions than
Chapter 4
in the cerebellum. The initial high amount of radioactivity was cleared from the brain
within 30 min.
Figure 6. Two series of horizontal (ventral to dorsal) PET images of two Wistar rats
(head) injected with 26.9 MBq ((0.23 nmol) of [18F]-FDEGPECO under baseline
conditions (A) and 20.5 MBq (0.26 nmol) of [18F]-FDEGPECO under blocking
conditions (B). Regions of interest were marked as HG (Harderian glands), fb (forebrain
containing striatum, hippocampus and cortex) and Ce (cerebellum). Raw data were
reconstructed and summed from 0 to 30 min post injection.
Further PET experiments were performed in two rats in order to analyze the specificity of
forebrain uptake of [18F]-FDEGPECO. One Wistar rat was scanned under baseline
conditions and the other under blockade conditions. Brain regions such as striatum,
hippocampus and cortex were only visualized under baseline conditions (Fig. 6A). Coadministration of M-MPEP (1mg/kg) with the radiotracer resulted in blockade of the
forebrain signal (Fig. 6B). Apart from specific [18F]-FDEGPECO signals in the forebrain,
PET images were characterized by non-specific tracer accumulation in the Harderian
glands.
Post mortem biodistribution studies with [18F]-FDEGPECO under control and blockade
conditions were carried out. Under control conditions, the highest levels of radioactivity
accumulation in the brain were found in the striatum (0.18 ± 0.02%IDnorm./g),
hippocampus (0.16 ± 0.02%IDnorm./g) and the cortex (0.13 ± 0.01%IDnorm./g) while
the cerebellum and the midbrain exhibited the lowest uptake levels of 0.06 ±
0.0007%IDnorm./g and 0.02 ± 0.002%IDnorm./g, respectively (Fig.7A). The striatum-tocerebellum and the hippocampus-to-cerebellum ratios were 2.89 and 2.57, respectively.
While no significant blocking effect was observed for the cerebellum and midbrain
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130
regions, accumulation of activity in the striatum and the hippocampus was reduced to
0.09 ± 0.003%IDnorm./g and 0.08 ± 0.005%IDnorm./g, which corresponds to a decrease
of 50%. The reduction in uptake of radioactivity in the cortical regions was in the same
range. For all the peripheral organs examined, the liver displayed the highest
accumulation of radioactivity (Fig. 7B). Kidney uptake was low and amounted to 0.11 ±
0.003%IDnorm./g. As expected, blocking with M-MPEP did not result in any significant
decrease of tracer uptake in the peripheral organs (Fig. 7B).
Figure 7. Biodistribution of [18F]-FDEGPECO in selected brain regions (A) and peripheral
organs (B). Animals were sacrificed 15 min post injection. The blockade group (right
columns) received co-injection of M-MPEP (1 mg/kg) and [18F]-FDEGPECO; the control
group (left columns) received 18F]-FDEGPECO and vehicle (PEG200/water = 1:1). Data are
presented as the mean of the percentage of normalized injected dose per gram of tissue ±
standard deviation (n = 3).
Chapter 4
4.5 Discussion
We recently published the successful fluorine-18 radiolabeling and the in vitro binding
affinity of [18F]-FDEGPECO (Ki = 3.8 ± 0.4 nM, KD1 = 0.6 ± 0.2 and KD2 = 13.7 ± 4.7
nM)18. During this study the radiochemical yield of this tracer was improved from 35% to
50%. This improvement was achieved through an improved purification step and a
shorter total synthesis time. High specific activities ranging from 80 to 320 GBq/µmol
were obtained.
[18F]-FDEGPECO was characterized in vitro for its stability in plasma and in human
microsomes. The stability studies indicated that [18F]-FDEGPECO is metabolically stable
since no radioactive degradation products were detected in vitro. P-gp is one important
carrier system that is responsible for the transportation of drugs out of the brain which
affects the pharmacokinetic profile of drugs or PET tracers severely27, 28. Thus, in order to
verify whether [18F]-FDEGPECO is not a P-gp substrate, an in vitro investigation of the
radioligand in a P-glycoprotein substrate test was undertaken. The relatively high
permeation rate from apical to basal, i.e. from luminal to abluminal, together with the
moderate lipophilicity of [18F]-FDEGPECO (logDpH7.4 = 1.7) suggests free diffusion of
the compound across the blood-brain barrier. Interestingly, apical to basal permeation of
[18F]-FDEGPECO was higher than basal to apical permeation across the hMDR1-MDCK
cells. We can thus with certainty establish that [18F]-FDEGPECO is not a P-gp substrate.
In vitro autoradiographical studies on rat brain slices with [18F]-FDEGPECO resulted in a
heterogeneous and displaceable uptake of activity in mGluR5-rich brain regions. As
expected, the highest amount of activity was found in mGluR5-rich brain regions such as
hippocampus, striatum and cortex (Fig. 3A). Accumulation of radioactivity in the
cerebellum was negligible. The substantially reduced and homogenous accumulation of
radioactivity observed under blocking conditions using ABP688 demonstrated the
specificity of [18F]-FDEGPECO for mGluR5 in vitro.
In order to evaluate the in vivo imaging characteristics and to elucidate how far the in
vitro data corresponded to the in vivo situation, PET studies in Wistar rats were
performed with [18F]-FDEGPECO. As observed in the autoradiographical studies, PET
imaging with [18F]-FDEGPECO also revealed a heterogeneous radioactivity accumulation
in mGluR5-rich brain regions such as hippocampus, striatum and cortex. Low levels of
radioactivity uptake were observed for the cerebellum, a brain region with a low
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132
expression level of mGluR5 (Fig. 4 and 6A). Thus, the distribution of [18F]-FDEGPECO
in the rat brain is in agreement with the expression pattern of mGluR5 in the mammalian
brain29. Blocking studies by co-injection of [18F]-FDEGPECO and M-MPEP (1 mg/kg),
the well known specific antagonist for mGluR530, caused a significant reduction in
radioactivity accumulation in mGluR5-rich brain regions. This indicates again the
specificity of the tracer for mGluR5 and is in line with the results of the in vitro
autoradiography. However, a large amount of unspecific uptake of radioactivity was
observed for the Harderian glands (Fig. 4 and 6A), a characteristic that has been described
for CNS PET tracers such as [18F]-fallypride22. The impact of this accumulation in
neighboring regions is unclear. The PET images showed no accumulation of radioactivity
in the bones or the skull, which points to the in vivo stability of the carbon-fluorine bond
in [18F]-FDEGPECO.
The time-activity curves (TACs) of the mGluR5-rich brain regions such as hippocampus,
striatum and cortex that were summarized as one region of interest (ROI) and the TAC of
the cerebellum displays besides the pattern of radioactivity accumulation, the
pharmacokinetic profile of [18F]-FDEGPECO in the rat brain (Fig. 5). The tracer cleared
quickly from the brain and has therefore a relatively short retention time in brain tissue
(t1/2 approximately 10 min). The obtained PET images of [18F]-FDEGPECO in rats
therefore were of lower quality compared to those acquired with [11C]-ABP68831.
The biodistribution studies in Wistar rats were also performed under control and blockade
conditions. The sacrifice time point of 15 min post injection was chosen based on the
time-activity curves of the PET experiments. The dissection studies also confirmed the
specificity of radioligand uptake in mGluR5-rich brain regions. A 50% blocking effect
was observed when the radiotracer was co-injected with M-MPEP (1 mg/kg). In the
cerebellum, where mGluR5 expression is lowest, no reduction in radioactivity uptake was
observed under blockade conditions. In a preliminary ex vivo metabolite analysis of rat
brain extracts performed at 5 and 15 min post injection, we identified besides the
radiolabeled parent compound, a more hydrophilic radioactive metabolite which accounts
for 7% of the total radioactivity (data not shown). Presumably, such hydrophilic
degradation products are structurally different from mGluR5 ligands and thus bind nonspecifically. Although this hydrophilic radiometabolite occurs in low amounts, it may
confound the signal and perhaps contribute to the remaining radioactivity not blocked
under blockade conditions. Certainly this may not be the only reason. Other factors such
Chapter 4
as non-specific binding of [18F]-FDEGPECO may also play a role. The lack of
radiodefluorination arising from the high stability of the C-F bond, the slightly more
favorable kinetic profile as well as the more favorable uptake ratios of [18F]-FDEGPECO
demonstrate that this novel tracer is superior to previously described [18F]-labeled
ABP688-analogues.
In conclusion, [18F]-FDEGPECO represents a new fluorinated ABP688 derivative with
excellent in vitro characteristics. The PET studies with [18F]-FDEGPECO allowed the
visualization of mGluR5 in the rat brain. The imaging of mGluR5 with [18F]-FDEGPECO
in humans may be a further step forward in tracer evaluation as a different
pharmacokinetic profile in humans might yield PET images of even higher quality due to
the stronger and longer-lived signals.
Acknowledgment
We acknowledge the technical support of Petra Wirth, Claudia Keller, Cindy Fischer and
Mathias Nobst and we thank Harriet Struthers for fruitful discussions.
4.6
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Conclusion and Future Perspectives
138
Conclusion and Further Directions
139
Conclusion and Future Perspectives
The development of [11C]-ABP688 laid the foundation for the non-invasive in vivo
imaging of the metabotropic glutamate receptor subtype 5 (mGluR5) in rats and humans.
However, the wide-spread use of this excellent imaging agent is limited to PET centers
with a cyclotron and radiochemistry facility due to the short physical half life of carbon11 (t1/2 = 20 min). Therefore, tremendous effort has been spent on the development of a
fluorine-18 labeled PET tracer during the last years. The aim of this project was to
develop a high-affinity and selective fluorine-18 labeled derivative of ABP688 that is
synthetically easily accessible as well as metabolically stable.
In this thesis seventeen ABP688 derivatives were successfully synthesized and evaluated
for their binding affinity to mGluR5. Six fluorine containing analogues of ABP688
exhibited high binding affinity to mGluR5 with Ki values below 10 nM. Two of the
analogues, FTECMO and FDEGPECO had outstanding in vitro properties and were
evaluated for their suitability as mGluR5 PET imaging agents. [18F]-FTECMO, a thiazole
analogue of ABP688, failed in vivo due to radiodefluorination. The second PET tracer
candidate, [18F]-FDEGPECO, exhibited excellent in vitro properties and 50% specificity
in vivo in rats. However, [18F]-FDEGPECO has two shortcomings that could limit its
further use: (1) fast clearance from mGluR5 regions and (2) a low striatal to cerebellar
uptake ratio. The newly developed tracers do not excel the existing [18F]-labeled
compounds such as [18F]SP203 and [18F]F-PEB. Therefore, the search for new mGluR5
PET ligands should aim to improve on the disadvantages of [18F]-FTECMO and [18F]FDEGPECO.
Figure 1. Structures of mGluR5 ligands.
One compound, (E)-3-(pyridin-2-ylethynyl)cyclohex-2-enone O-3-fluoropyridin-2-yl
oxime (mFPPECO, Fig. 1.), has recently been identified. This compound bears a fluorine
atom in position 3 of the pyridine ring connected to the oxime moiety where the fluorine
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is expected to be stable under in vivo conditions unlike previously investigated 6fluoropyridine derivatives. With a high binding affinity (Ki = 4 nM) to mGluR5,
mFPPECO is worth developing. As much as a high in vivo stability is anticipated, the
development of a single-step radiosynthetic procedure for the labeling of this candidate
would be radiochemically challenging. Nevertheless, the evaluation of this candidate is
recommended due to its high binding affinity and convenient lipophilicity (clogP = 2.5).
The introduction of a fluorine atom at the cyclohexenone moiety of the ABP688 molecule
has not yet been pursued. This approach, however, will lead to relatively complicated
chemistry due to the presence of enantiomers after fluorine atom introduction. This
problem might be overcome with enantioselective syntheses. Few more fluorine
containing analogues of ABP688 are also imaginable. Another proposal is to develop
compounds that are structurally unrelated to ABP688. A possible future lead compound,
fenobam, is depicted in Figure 5.1.
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Curriculum Vitae
Cindy Anna Baumann
Born on the 24th of August 1981 in Amberg (Bavaria)
Education
2007 – 2010
2006
ETH Zurich, Institute for Pharmaceutical Sciences,
Radiopharmaceutical Sciences
PhD thesis: Development of Novel Fluorine-18 Labeled PET
Tracers for Imaging the Metabotropic Glutamate Receptor
Subtype 5(mGluR5)
Licensed Pharmacist (Approbation) in Germany
2005 – 2006
Practical training as part of pharmaceutical education
2004
ETH Zurich, Institute of Pharmaceutical Sciences,
Internship in Drug Formulation & Delivery
2001 – 2005
University of Regensburg, Institute of Pharmacy
Studies of Pharmaceutical Sciences
1993 – 2001
Dr.-Johanna-Decker Schulen von Unserer Lieben Frau
Abitur 2001 (1.0)
Employment
2007 – 2010
2006
2005-2006
PhD student in the group of Professor Pius August Schubiger,
Swiss Federal Institute of Technology (ETH) Zurich
Pharmaceutical Trainee in the Parenterals Production of
F. Hoffmann-LaRoche in Basel
Pharmaceutical Trainee in the Raphael Apotheke, Lindenberg
Courses
2007 - 2010
International PhD Program in Neuroscience at the
Center for Neurosciences Zurich
2009
Bayer PhD Student Course, Cologne
2008
English Scientific Writing Course,
Teaching Course
Didaktikzentrum ETHZ
2008
Animal Handling Course, (TK I), ETHZ
2008
Radiation Safty Course (Strahlenschutzsachbeauftragte)
Paul-Scherrer Institute (PSI) Villigen
142
143
Publications
Structure-Activity Relationships of Fluorinated ABP688 Derivatives and the
Discovery of a High Affinity Analogue as a Potential Candidate for Imaging
mGluR5 with PET
Cindy A. Baumann, Linjing Mu, Sinja Johannsen, Michael Honer, Pius A. Schubiger,
Simon M. Ametamey
Journal of Medicinal Chemistry, submitted
Syntheses and Pharmacological Characterization of Novel Thiazole Derivatives as
Potential MGluR5 PET Ligands
Cindy A. Baumann, Linjing Mu, Nicole Wertli, Michael Honer,Pius A. Schubiger,
Simon M. Ametamey
Journal of Bioorganic and Medicinal Chemistry, submitted
In vitro and in vivo Evaluation of [18F]-FDEGPECO as Novel Fluorine-18 Labeled
PET Tracer for Imaging Metabotropic Glutamate Receptors Subtype 5
Cindy A. Baumann, Linjing Mu, Michael Honer, Sara Belli, Stefanie-Dorothea Krämer,
Pius. A. Schubiger, Simon M. Ametamey
Neuroimage, prepared for submission
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Oral Presentations
Development of novel fluorine-18 labelled PET tracers for imaging the metabotropic
glutamate receptor subtype 5 (mGluR5)
EANM'09 Annual Congress of the European Association of Nuclear Medicine ,
Barcelona, October 10 – 14 2009
Development of novel fluorine-18 labelled PET tracers for imaging the metabotropic
glutamate receptor subtype 5 (mGluR5)
SGR-SSR- SGNM (Schweizerische Gesellschaft für Nuklearmedizin) Kongress, Geneva,
Switzerland, June 2 – 6 2009
Development of novel fluorine-18 labeled PET tracers for imaging the metabotropic
glutamate receptor subtype 5 (mGluR5)
IPW Doktorandentag, ETH Zurich, 9 Februar 2009
Development of novel fluorine-18 labeled PET tracers for imaging the metabotropic
glutamate receptor subtype 5 (mGluR5)
16. Arbeitstagung der AG Radiochemie/Radiopharmazie der DGN, Münster, Germany,
25-27 September 2008
145
Poster Presentations
Development of novel fluorine-18 labeled PET tracers for imaging the metabotropic
glutamate receptor subtype 5 (mGluR5)
18th International Symposium on Radiopharmaceutical Sciences meeting, Edmonton,
Canada, 12-17 July 2009
Development of novel fluorine-18 labeled PET tracers for imaging the metabotropic
glutamate receptor subtype 5 (mGluR5)
ZNZ PhD retreat, Valens, Switzerland, 21-23 May 2009
Log P value as important factor for the development of fluorine-18 labeled PET
tracers for imaging of metabotropic glutamate receptors subtype 5 (mGluR5) in the
CNS
The 4th LogP Symposium, ETH Zurich, Switzerland, 8-11 February 2009
Development of fluorine-18 labeled radioracers for PET imaging of metabotropic
glutamate receptors subtype 5 (mGluR5)
ZNZ Symposium 2008, Zurich, Switzerland, 12 September 2008
Development of improved radiotracers for PET imaging of metabotropic glutamate
receptors subtype 5 (mGluR5)
6th FENS Forum of European Neuroscience, Geneva, Switzerland, 12 – 16 July, 2008
Development of novel PET tracers for the imaging of metabotropic glutamate
receptors subtype 5
ZNZ Symposium 2007, Zurich, Switzerland, 14 September, 2007
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Awards
John Wiley & Sons 2009 JLCR Young Scientists Prize
18th International Symposium on Radiopharmaceutical Sciences,
Edmonton, Canada
12-17th July 2009
Dr. Siegfried Award for the best oral presentation
Doktorandentag, ETHZ
Zurich
11th Feb 2009