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. 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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. 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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 References 1. Andreas Ritzén, J. M. M., Christian Thomsen,, Molecular Pharmacology and Therapeutic Prospects of Metabotropic Glutamate Receptor Allosteric Modulators. Basic Clin. Pharmacol. 2005, 97 (4), 202-213. 2. Conn, P. 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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. 125 126 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. 127 128 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 129 130 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 131 132 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 References 1. 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J.; Gasparini, F.; Kuhn, R., The Non-competitive Antagonists 2-Methyl-6(phenylethynyl)pyridine and 7-Hydroxyiminocyclopropan[b]chromen-1a- carboxylic Acid Ethyl Ester Interact with Overlapping Binding Pockets in the Transmembrane Region of Group I Metabotropic Glutamate Receptors. J. Biol. Chem. 2000, 275 (43), 33750-33758. 31. Ametamey, S. M.; Kessler, L. J.; Honer, M.; Wyss, M. T.; Buck, A.; Hintermann, S.; 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. 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 140 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. 141 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 144 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 146 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
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