Exploring Palladium-Mediated 11C/12C-Carbonylation Reactions

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 241

PET Tracer Development Targeting the Vesicular Acetylcholine Transporter

SARA ROSLIN

ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6192 ISBN 978-91-513-0136-5 UPPSALA urn:nbn:se:uu:diva-332359 2017 Dissertation presented at Uppsala University to be publicly examined in Hall B:21, BMC, Husargatan 3, Uppsala, Friday, 15 December 2017 at 09:15 for the degree of Doctor of Philosophy (Faculty of Pharmacy). The examination will be conducted in English. Faculty examiner: Ph.D. Victor Pike (National Institute of Mental Health, Molecular Imaging Branch).

Abstract Roslin, S. 2017. Exploring Palladium-Mediated 11C/12C-Carbonylation Reactions. PET Tracer Development Targeting the Vesicular Acetylcholine Transporter. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 241. 99 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0136-5.

The work presented herein describes the utilization and exploration of palladium-mediated incorporations of carbon monoxide and/or [11C]carbon monoxide into compounds and structural motifs with biological relevance. The first part of the thesis describes the design, synthesis and 11C-labeling of prospective PET tracers for the vesicular acetylcholine transporter (VAChT), a target affected in several neurodegenerative diseases. Different parts of the benzovesamicol scaffold were modified in papers I and II to probe the binding to VAChT. The key motif was an amide functional group, which enabled the use of palladium-mediated 11C/12C-carbonylations to synthesize and evaluate two different sets of structurally related ligands. The second part of the thesis describes the exploration of different aspects of palladium- mediated 11C/12C-carbonylation reactions. The utilization of unactivated alkyl iodides and bromides as coupling partners in a carbonylative Suzuki-Miyaura reaction was described in paper III. The combination of palladium-catalysis together with visible light irradiation enabled their functionalization via an alkyl radical. The mild conditions, namely the ambient temperature and pressure of carbon monoxide, and the accessible reaction set-up further added to the utility of the method. A palladium(II)-mediated oxidative 11C-carbonylation for synthesis of 11C-labeled ureas was described in paper IV. Utilizing only amines in addition to a palladium-source and [11C]carbon monoxide, the method proved to be facile and robust, thus representing a simplification in relation to methods using other 11C-synthons for synthesis of 11C-labeled ureas. Finally, a palladium(0)-catalyzed carbonylation reaction for synthesis of acylamidines was presented in paper V. The versatility of the method was demonstrated by one-pot cyclizations to form oxadiazoles and triazoles together with the corresponding 11C-carbonylation reaction to produce 11C-labeled acylamidines and an oxadiazole. The work described herein has thus contributed structural information in the search for a PET tracer for VAChT and identified a viable lead structure for future investigations. Furthermore, investigation of reaction conditions that would allow use of either elusive or accessible substrates led to the development of methods for synthesis and/or 11C-labeling of various carbonylated compounds.

Keywords: Carbonylation, palladium, carbon-11, radiochemistry, positron emission tomography, vesicular acetylcholine transporter, vesamicol, alkyl halide, oxidative carbonylation, acylamidine, oxadiazole, heterocycle

Sara Roslin, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry, Box 574, Uppsala University, SE-75123 Uppsala, Sweden.

ISSN 1651-6192 ISBN 978-91-513-0136-5 urn:nbn:se:uu:diva-332359 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-332359)

Till min växande familj

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Bergman, S., Estrada, S., Hall, H., Rahman, R., Blomgren, A., Larhed, M., Svedberg, M., Thibblin, A., Wångsell, F., Antoni, G. (2014) Synthesis and Labelling of a Piperazine-Based Library of 11C-Labeled Ligands for Imaging of the Vesicular Acetylcholine Transporter. Journal of Labelled Compounds and Radiopharma- ceuticals, 57(8):525–532. II Roslin, S., De Rosa, M., Deuther-Conrad, W., Eriksson, J., Odell, L.R., Antoni, G., Brust, P., Larhed, M. (2017) Synthesis and In Vitro Evaluation of 5-Substituted Benzovesamicol Ana- logs containing N-Substituted Amides as Potential Positron Emission Tomography Tracers for the Vesicular Acetylcholine Transporter. Bioorganic & Medicinal Chemistry, 25(19):5095– 5106. Part of special issue “Design and Synthesis of Bioactive Compounds”. III Roslin, S., Odell, L.R. (2017) Palladium and Visible-Light Me- diated Carbonylative Suzuki-Miyaura Coupling of Unactivated Alkyl Halides and Aryl Boronic Acids. Chemical Communica- tions, 53:6895–6898. IV Roslin, S., Brandt, P., Nordeman, P., Larhed, M., Odell, L.R., Eriksson, J. (2017) Synthesis of 11C-Labelled Ureas by Palla- dium(II)-Mediated Oxidative Carbonylation. Molecules, 22(10):1688. Part of special issue “Current Aspects of Radio- pharmaceutical Chemistry”. V Rydfjord, J., Roslin, S., Roy, T., Abbas, A., Stevens, M., Larhed, M., Odell, L.R. Acylamidines by Pd-Catalyzed Aminocarbonyl- ation: One-Pot Cyclizations and 11C-Labeling. Manuscript.

Reprints were made with permission from the respective publishers.

Author Contribution Statement

The following contributions were made by the author of this thesis to each paper:

I Synthesized reference compounds and starting material for 11C- labeling experiments, prepared 11C-labeled compounds, collated experimental data and drafted the manuscript. II Designed and synthesized compounds for affinity determinations and synthesized starting material for 11C-labeling experiments, performed 11C-labeling experiments, collated experimental data and drafted the manuscript. III Designed and carried out method development, designed and synthesized starting materials and final compounds, collated experimental data and drafted the manuscript. IV Designed and carried out method development, designed and synthesized starting material and reference compounds, performed 11C-labeling experiments, collated data and drafted the manuscript. V Performed 11C-labeling experiments, assisted in collating experimental data and manuscript preparation.

Papers Not Included in This Thesis

Lampa, A.K., Bergman, S.M., Gustafsson, S.S., Alogheli, H., Åkerblom, E.B., Lindeberg, G.G., Svensson, R.M., Artursson, P., Danielson, U.H., Kar- lén, A., Sandström, A. (2014) Novel Peptidomimetic Hepatitis C Virus NS3/4A Protease Inhibitors Spanning the P2-P1’-region. ACS Medicinal Chemistry Letters, 5(3): 249–254.

Chow, S.Y., Stevens, M.Y., Åkerbladh, L., Bergman, S., Odell, L.R. (2016) Mild and Low-Pressure fac-Ir(ppy)3-Mediated Radical Aminocarbonylation of Unactivated Alkyl Iodides through Visible-Light Photoredox Catalysis. Chemistry – A European Journal, 22(27):9155–9161. Hot Paper. Featured on the cover of the July 2016 issue.

Roslin, S., Odell, L.R. (2017) Visible-Light Photocatalysis as an Enabling Tool for the Functionalization of Unactivated C(sp3)-Substrates. European Journal of Organic Chemistry, 2017(15):1993–2007. Part of Special issue: Photoredox Catalysis.

Contents

Thesis Overview ...... 15 Introduction ...... 17 Palladium Catalysis ...... 17 The Palladium Mediated Carbonylation Reaction ...... 18 Positron Emission Tomography and Tracer Development ...... 21 Positron Emission Tomography ...... 21 Considerations in Tracer Development ...... 23 11C-Carbonylative Radiochemistry ...... 26 Targeting the Cholinergic System ...... 29 The Cholinergic System ...... 29 The Cholinergic System in Neurodegenerative Diseases ...... 30 The Vesicular Acetylcholine Transporter ...... 32 Development of Ligands for the Vesicular Acetylcholine Transporter ...... 32 Design, Synthesis and 11C-Labeling of Ligands for the Development of a PET Tracer for VAChT ...... 36 Synthesis and Labelling of a Piperazine-Based Library of 11C-Labeled Ligands for Imaging of the Vesicular Acetylcholine Transporter (Paper I) ...... 37 Aim ...... 37 Design, Synthesis and 11C-Labeling ...... 3 7 Preclinical evaluation...... 39 Synthesis and In Vitro Evaluation of 5-Substituted Benzovesamicol Analogs containing N-Substituted Amides as Potential Positron Emission Tomography Tracers for the Vesicular Acetylcholine Transporter (Paper II) ...... 41 Aim ...... 41 Design and Synthesis of 5-Substituted Benzovesamicol Analogs containing N-Substituted Amides ...... 42 In Vitro Evaluation ...... 43 11C-Aminocarbonylation ...... 45 Summary of Papers I and II ...... 47

Method Development: Palladium-Mediated Incorporation of [11C]Carbon Monoxide or Carbon Monoxide ...... 49 Palladium and Visible-Light Mediated Carbonylative Suzuki-Miyaura Coupling of Unactivated Alkyl Halides and Aryl Boronic Acids (Paper III) ...... 49 Aim ...... 50 Optimization of Reaction Conditions ...... 50 Investigation of the Reaction Scope ...... 52 Proposed Catalytic Cycle ...... 55 Synthesis of 11C-Labelled Ureas by Palladium(II)-Mediated Oxidative Carbonylation (Paper IV) ...... 57 Aim ...... 58 Synthesis of 11C-Labeled N,N’-Disubstituted Ureas ...... 58 Synthesis of 11C-Labeled N,N’,N’-Trisubstituted Ureas ...... 60 Proposed Reaction Mechanism ...... 64 Acylamidines by Pd-Catalyzed Aminocarbonylation: One-Pot Cyclizations and 11C-Labeling (Paper V) ...... 66 Aim ...... 67 Acylamidine Synthesis and One-Pot Cyclizations ...... 67 11C-Labeled Acylamidines and One-Pot Cyclization ...... 69 Summary of Papers III–V ...... 71 Concluding Remarks ...... 73 Acknowledgments...... 75 Appendix 1 ...... 78 Small Animal PET and Biodistribution Studies (Paper I) ...... 78 Small Animal PET ...... 78 Organ distribution studies ...... 78 List of References ...... 80

Calculations and Definitions

Labeled compound: A labeled compound is a mixture of an isotopically unmodified compound and one (or more) analogous isotopically substituted compound(s). Square brackets are used to indicate the substituted isotope, e.g. [11C]carbon monoxide.

Hot cell: A radiation-shielded laboratory unit where radiochemistry is performed.

Decay-corrected: Calculations using activities measured at different time- points but corrected to the same point in time are referred to as decay-cor- -λt rected. The formula used is At = A0e , where λ = ln 2/t1/2 and t1/2 is the half- life of the radionuclide.

[11C]CO-conversion: The fraction of [11C]carbon monoxide converted to non- volatile 11C-labeled products (referred to as Trapping Efficiency in paper I). Decay-corrected.

Product selectivity: The fraction of 11C-labeled product formed in the reaction, based on analytical HPLC of crude reaction mixture.

Radiochemical yield (RCY): Based on the starting radioactivity and the radioactivity of the 11C-labeled product isolated after semi-preparative HPLC. De- cay-corrected. An estimate of the radiochemical yield for the non-isolated 11C- labeled product is based on the [11C]CO-conversion and the product selectivity. Used when optimizing 11C-labeling reactions.

Identification of 11C-labeled product: The identity of the 11C-labeled product is confirmed by addition of the isotopically unmodified product to an aliquot of the product, followed by HPLC analysis and comparison of retention times of the UV peak and the radio peak.

11 Molar activity (Am): The radioactivity of the isolated C-labeled product measured at a defined point in time, divided by the molar amount of the product. Commonly expressed in GBq/µmol.

Radiochemical Purity (RCP): The fraction of 11C-labeled product in the isolated product fraction.

Abbreviations

ABV 5-Aminobenzovesamicol ACh Acetylcholine AChE Acetylcholinesterase AD Alzheimer’s disease Am Molar activity 1-Cbz-piperazine Benzyl piperazine-1-carboxylate ChAT Choline acetyltransferase CNS Central nervous system CO Carbon monoxide DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene DFT Density functional theory DIAD Diisopropyl azodicarboxylate DIPEA N,N-Diisopropylethylamine DMAP 4-(Dimethylamino)pyridine DMF N,N-Dimethylformamide DPEphos (Oxydi-2,1-phenylene)bis(diphe- nylphosphine) Dppf 1,1’-Bis(diphenylphosphino)ferrocene Dppp 1,3-Bis(diphenylphosphino)propane DTG 1,3-Di-o-tolylguanidine EOB End of bombardment 2 Fac-Ir(ppy)3 Tris[2-phenylpyridinato-C ,N]iridium(III) [18F]FEOBV (–)-5-[18F]Fluoroethoxybenzo- vesamicol HBTU N,N,N′,N′-Tetramethyl-O-(1H-benzo- triazol-1-yl)uranium hexafluorophos- phate HE Hantzsch ester HPLC High-performance liquid chromatog- raphy Kd Dissociation constant Ki Inhibition constant LED Light-emitting diode mCPBA meta-Chloroperoxy benzoic acid

NHP Non-human primate PET Positron emission tomography pKa Acid dissociation constant PPh3 Triphenylphosphine P(o-tol)3 Tris(o-tolyl)phosphine RCP Radiochemical purity RCY Radiochemical yield sEH Soluble epoxide hydrolase SET Single electron transfer σ (σ1 and σ2) Sigma receptors (sigma-1 and sigma- 2) SPECT Single-photon emission computed tomography SUV Standardized uptake value TBAI Tetrabutylammonium iodide TEMPO 2,2,6,6-Tetramethylpiperidine-1-oxyl TFA Trifluoroacetic acid THF Tetrahydrofuran Triflate Trifluoromethanesulfonate TS Transition state VAChT Vesicular acetylcholine transporter Xantphos 4,5-Bis(diphenylphosphino)-9,9-di- methylxanthene XPhos 2-Dicyclohexylphosphino-2′,4′,6′-tri- isopropylbiphenyl

Thesis Overview

Many reactions sort under the term “carbonylation reaction”. In common they have the possibility of joining either simple or more complex starting materials to form elaborate carbonylated structures and the possibility of incorporat- ing isotopically modified carbon monoxide into the compound of interest. The work presented in this thesis has focused on palladium-mediated incorporation of carbon monoxide or [11C]carbon monoxide into compounds of biological relevance. The main part of this thesis is dedicated to radiochemistry and its application in positron emission tomography (PET), a highly valuable imaging modality utilizing molecular probes (tracers) labeled with a positron-emitting radionuclide such as carbon-11. To take full benefit of PET and to further advance PET, there is a need to develop new tracers to add to the arsenal of existing PET tracers. Similarly, there is a need to develop methods for the incorporation of the positron-emitting radionuclide into biologically relevant, tracer-like molecules, to add to the radiochemical toolbox. A smaller part of the thesis is dedicated to exploration of different aspects of palladium- catalyzed carbonylation reactions, such as substrate and product scopes as well as reaction conditions. However, the general features of carbonylation reactions allow these parts to overlap. Thus, 11C- and 12C-carbonylation reactions have been utilized as tools to enable the synthesis and evaluation of aspiring tracers for the vesicular acetylcholine transporter, a target affected in several neurodegenerative diseases (papers I and II). Palladium-mediated carbonylation reactions have been explored in the development of a method for utilization of challenging, unactivated alkyl halides in a carbonylative Suzuki-Miyaura coupling (paper III) and in the development of methods for the incorporation of carbon monoxide or [11C]carbon monoxide into ureas, acylamidines and heterocycles (papers IV and V).

Introduction

Palladium Catalysis Palladium is one of the most widely employed transition metals in organic synthesis. In the late 1950s, with the Wacker process, palladiums utility in synthetic organic chemistry began to be appreciated.1 Today, palladium catalysis is a routine feature in organic synthesis because of its versatility and the selectivity that can be achieved, both on the laboratory scale and on the industrial scale. The importance of palladium catalysis in organic chemistry was recognized in 2010, when the Nobel Prize in Chemistry was awarded to Ei- ichi Negishi, Richard F. Heck and Akira Suzuki for their contributions to the field of palladium-catalyzed cross-couplings.2 These, and other important palladium(0)-catalyzed cross-couplings are depicted in Figure 1.

Figure 1. Selected palladium(0)-catalyzed cross-couplings.3–8

The catalytic activity of transition metals largely stems from their electronic configuration and their empty d-orbitals to which they can accept electrons from ligands, thus forming transition metal complexes. A ligand can be a solvent molecule, a base, a reactant or an additive.9 The catalytic activity is also dependent on the ability of the complex to change from a stable to a reactive complex and that the interconversion of the two complexes is kinetically facile under the applied reaction conditions. Palladium preferably exists in the 0 and +2 oxidation states, with d10 and d8 electronic configurations, respectively.

17 There is a narrow energy gap between these two states, making palladium an excellent catalyst for two-electron processes such as oxidative addition and reductive elimination. This tendency for two-electron processes is believed to make palladium less prone to undergo one-electron processes as side-reactions although there are exceptions.10–14 The reactivity of palladium is further related to the ability to form coordinatively unsaturated complexes, thereby providing one or more free coordination sites for interaction with substrates. The relatively high electronegativity of palladium (2.2) also affects the reactivity and renders the Pd-C bond rather non-polar in contrast to organomagne- sium (Grignard) reagents or organolithium reagents, wherein the metal-carbon bonds are highly polarized. This difference makes the reactivity of organopalladium complexes largely orthogonal to that of the Grignard and organolithium reagents. Thus, the versatility and selectivity of palladium is based on these inherent properties but the reactivity of palladium can be tuned by selecting ligands with suitable electronic and steric properties.15–17

The Palladium Mediated Carbonylation Reaction The transition-metal catalyzed introduction of carbon monoxide into organic molecules was pioneered in the 1930s by Otto Roelen and Walter Reppe, by their work on the hydroformylation reaction and the hydrocarboxylation reaction.18 The reactions both featured unsaturated starting materials, metal catalysis and carbon monoxide and Walter Reppe coined the term “carbonylation” to describe the reactions. From 1968 and onwards, Richard Heck published a series of papers dis- closing a three-component carbonylation reaction, consisting of an electrophile, a nucleophile and carbon monoxide, assembled under palladium catalysis (Figure 2).19–22 Since the seminal work of Heck et al., impressive progress has been made in terms of substrate scope, choice of catalyst and source of carbon monoxide.23–27

Figure 2. Basic constituents of the (Heck) carbonylation reaction.

The electrophilic component is often a (hetero)aryl, alkenyl or benzyl halide or pseudohalide.23,24 The use of alkyl substrates have been limited by their relatively slow oxidative addition to the palladium(0) catalyst and the fact that they are prone to undergo beta elimination from the σ-alkylpalladium complex formed.26,28,29 The rate of the oxidative addition is largely influenced by the C-

18 X bond.24 The weaker the bond, the more reactive the substrate is toward oxidative addition in the order C-I > C-OTf ≥ C-Br > C-Cl. The nucleophilic component can either be an amine, alcohol, thiol or an organometallic reagent.24 The interest in the carbonylation reaction lies in the existence and utilization of carbonyl containing derivatives in natural products, pharmaceutical products, industrial products and in other fields. An advantage of the carbonylation reaction is the ability to utilize isotopically modified carbon monoxide ([11C]CO, [13]CO and [14C]CO).30–32 Carbon monoxide is thermally stable whilst chemically reactive to transition metals and is thus viewed as a convenient one-carbon building block despite its inherent toxicity and flammability. These negative features of carbon monoxide demand special attention and equipment, which is why the use of alternative non-gaseous sources of carbon monoxide, such as molybdenum hexacarbonyl (Mo(CO)6), has gained interest.27,33 A generic catalytic cycle for a palladium(0)-catalyzed carbonylation of an aryl halide is depicted in Figure 3.

Figure 3. A palladium-catalyzed carbonylation reaction, with a heteroatom-based nucleophile (I-VI) or an organometallic reagent (I-IV, VII-VIII).

The main features are formation of the active palladium catalyst, a 14-electron palladium(0) species, from a palladium(0) or palladium(II) source (I).34 Oxi- dative addition of the electrophile forms a σ-arylpalladium(II) complex (II).

19 Carbon monoxide will then coordinate to the palladium(II) complex (III) followed by 1,1-insertion of carbon monoxide into the aryl-palladium bond, thus forming an acyl-palladium(II) complex (IV). Nucleophilic attack on the acyl- palladium complex gives product (V) before a reductive elimination with a base regenerates the active palladium(0) complex (VI). In the case of organometallic reagents (M-Rˈ), a transmetallation to the acyl-palladium complex will occur (VII) before a reductive elimination furnishes the product and regenerates the palladium catalyst (VIII). Of consideration here is the ability of carbon monoxide to function as a ligand.35 Donation of an electron pair to an empty d-orbital on palladium results in the formation a σ-bond while donation of electron density from filled d-orbitals on palladium to an anti-bonding orbital (π*) on carbon monoxide results in so-called back bonding. The back bonding lowers the electron density on palladium, which can hamper the oxidative addition step.26,36 This may be problematic for alkyl halides, a substrate class for which oxidative addition is already challenged. As can be seen in Figure 3, the carbonylation reaction offers the possibility to reach complex products from (simple) starting materials by forming C-C or C-heteroatom bonds. Through variation of the starting materials, libraries of structurally related compounds can be built. The work presented in papers I- III and V features adaptations and new developments of the carbonylation reaction, in terms of substrates used and thus products formed as well as in the experimental set-up. In oxidative carbonylations, the carbonylated product is derived from the reaction of carbon monoxide with two nucleophiles.37–39 A palladium(II)-species is the active catalyst and the reaction therefore does not start with oxidative addition. During the reaction, palladium(II) is reduced to palladium(0) and a re-oxidant is needed to regenerate the active palladium catalyst. Nucle- ophiles can be either alkenes,40 hydrocarbons,41 amines41 or alcohols42 or various organometallic reagents such as boronate esters,43 thus generating carbonylated products such as carboxylic acids, lactames, ureas, carbonates and esters. An oxidative 11C-carbonylation of amines was explored in paper IV.

11C- and 12C-Carbonylation Reactions The sequence of events is the same for a carbonylation reaction with [11C]carbon monoxide and carbon monoxide but the low amount of [11C]carbon monoxide present in the 11C-reaction together with the short half-life of carbon-11 differentiates the conditions for the 11C- and 12C-carbonylation reactions. In the 11C-labeling reaction, the reactants and reagents are added in micromolar amounts whereas only nanomolar amounts of [11C]carbon monoxide are present. Thus, the substoichiometric amounts of [11C]carbon monoxide will render the reaction mediated by palladium as the metal will likely only react once rather than being catalyzed as in 12C-carbonylation reactions in

20 which substoichiometric amounts of palladium generate the product. The ob- ject in the 12C-carbonylation reaction then becomes to efficiently utilize the reagents to obtain full conversion of the organic substrates into product whereas the 11C-carbonylation reaction rather seeks to efficiently convert quantitative amounts of [11C]carbon monoxide into product. Here, the 12C- carbonylation reaction benefits from being able to adjust parameters such as carbon monoxide pressure and reaction time more readily. Therefore, in the 11C-carbonylation reaction, extra consideration should be given to the choice of palladium and ligand system, the leaving group on the electrophile and, if possible, the nucleophile. Andersen et al. approached this by utilizing pre- formed arylpalladium complexes in the 11C-carbonylative labeling of structurally challenging pharmaceuticals.44

Positron Emission Tomography and Tracer Development Positron Emission Tomography PET is a non-invasive imaging technique by which biochemical and physiological processes can be studied in vivo. The first PET camera for human use was constructed in 1971 and since then, PET has emerged as a prominent imaging modality in nuclear medicine and biomedical research.45 Clinical applications are mainly found in cardiology, neurology and oncology, with the latter comprising the majority of the PET studies conducted.46–48 Furthermore, the possibility to study pharmacological processes in vivo, such as receptor occupancy, metabolism and pharmacokinetic parameters, has established PET as a powerful technique in drug development.49–52 The basis for these intricate studies is the tracer and the tracer concept. The International Union of Pure and Applied Chemistry defines a tracer as a “for- eign substance mixed with or attached to a given substance to enable the distribution or location of the latter to be determined subsequently”.53 In nuclear medicine, a radioactive isotope has been incorporated into the tracer to allow for external detection. Here, the tracer can be either an endogenous compound 15 11 such as water ([ O]H2O) or an amino acid (e.g. [ C]methionine), a modified endogenous compound such as fluorodeoxyglucose ([18F]FDG), or a novel compound synthesized exclusively to study a certain process such as beta- amyloid tracer Pittsburgh compound B ([11C]PIB).54 The tracer concept was introduced by George de Hevesy and he was awarded the Nobel Prize in Chemistry in 1943 for his studies on chemical processes utilizing radioactive isotopes as tracers.55 The tracer concept can be described as the use of a tracer to follow and study biochemical and physiological processes at doses which do not elicit a response or in other ways affect the outcome of the process being studied.

21 In PET, the utilized radioactive isotopes are neutron deficient and decay mainly by positive beta decay, with the elementary process involving the transformation of a proton (p+) into a neutron (n) with the concomitant emis- + 56 sion of a positron (β ) and a neutrino (νe) (Equation 1). Some of these radio- isotopes also decay by electron capture in addition to the positive beta-decay however, electron capture only accounts for a comparatively small amount of the decay (Equation 2).

+ + p  n + β + νe (Equation 1) + - p + e  n + νe (Equation 2)

The positron gains kinetic energy in the decay, which causes it to travel a finite distance in tissue whilst losing energy to the surroundings.56,57 When essen- tially all of the kinetic energy has been lost, the positron will combine with an electron and annihilate. Two photons are formed in the annihilation event, each with an energy of 511 keV, and they are emitted at approximately 180° relative to each other. It is these photons and the collinearity of their emittance that is exploited in PET (Figure 4). By coincidence detection of the photons, signals are created and transformed into images showing radioactivity concentrations. PET is thus a highly sensitive imaging modality and provides high spatial resolution.

γ β+

Figure 4. Schematic depiction of a PET scan, here with an 11C-labeled tracer.

However, the resolution is lowered by phenomena that arise during a PET scan. The direction in which the positron is emitted is unpredictable and the energy with which the positron is emitted will vary, causing the distance trav- elled to vary. The angle which the photons are emitted at sometimes deviates

22 from 180° by ± 0.5° due to non-zero momentum in the annihilation event. Together with possible scattering of the photon inside the study subject, these phenomena lower the resolution possible to attain with PET. The data collected in the PET scan can be utilized for the detection of tu- mors and metastases, measurements of brain and myocardial blood flow or protein synthesis rates, for example.58 PET is often combined with either computed tomography (PET/CT) or magnetic resonance imaging (PET/MRI) to gain anatomical information in addition to the radioactivity concentration.59 The production of radionuclides utilized in PET requires a particle acceler- ator such as a cyclotron, with generator produced gallium-68 as an exception, and the characteristics of radionuclides frequently used in PET are summarized in Table 1.

Table 1. Characteristics of selected positron emitting radionuclides56,57,60 + Emax en- Mean β Theoretical Radio- Nuclear β+ emission t1/2 (min) ergy range molar activity nuclide (%) reaction (MeV) (mm)a (GBq/µmol) 11C 14N(p,α)11C 20.4 > 99 0.96 1.1 341 13N 16O(p,α)13N 10.0 > 99 1.2 1.5 699 15O 14N(d,n)15O 2.0 > 99 1.7 2.5 3394 18F 18O(p,n)18F 110 97 0.63 0.6 63 68Ga From 68Ge 68.3 89 1.9 2.9 102 aIn water.

Considerations in Tracer Development The development of a PET tracer is complex and requires a multidisciplinary approach. During the development process, several parameters need to be addressed, often in an iterative fashion. Identification of a viable target, the imaging of which will answer one or more biochemical questions, along with identification of a lead structure with affinity for the target, are natural starting points. Many of the parameters relate to the tracer concept, the ability to study a biological system without disturbing its function, and a number of important parameters are discussed below.

Radiochemistry The lead structure, and the derivatives synthesized, must be amenable for labeling with a positron-emitting radionuclide (Table 1). The choice of radionuclide to be incorporated should be guided by i) the half-life of the biological process under study which should be matched by the half-life of the radionuclide, ii) structural prerequisites of the compound/compounds to be labeled in relation to available labeled precursors, iii) the possibility to perform the labeling as a late step in the synthesis, as the time allowed for labeling together with the ensuing purification and formulation is dictated by the half-life of the

23 radionuclide. The low amount of the radionuclide produced affects the radiochemistry and the labeled precursor is thus found in substoichiometric amounts in relation to other reactants and reagents.

Specificity

The tracer must display a high affinity (Kd) for the target. How high the affinity need to be is dependent on the expression of the target in the particular tissue, measured as Bmax. The lower the Bmax, the higher the affinity needs to be to fulfill the tracer concept. The relationship between Bmax and the dissociation constant Kd is referred to as the binding potential, BP (BP = Bmax/Kd). The binding potential can thus be used as a guide for assessing the affinity needed. A BP > 10 can be regarded as a rule of thumb although the situation in vivo is much more complex and exceptions exist.61 While a tracer is required to bind specifically to the target, there is always a degree of non-specific binding, which lowers the signal-to-noise ratio. Un- like the saturable specific binding, the non-specific binding is non-saturable and could for example represent association with membrane structures as lip- ophilic molecules show a higher propensity for non-specific binding.

Selectivity The selectivity of a tracer is related to the binding of the tracer to other, off- target sites. A lack of selectivity will render ambiguous results, thus the aim should be for high selectivity (> 30-fold has been suggested).62 Selectivity is dependent on the relative affinities, tissue distribution and density of the respective target and off-target sites. Hence, a lower selectivity can be accepted if the off-target site is not co-located with the target or if the off-target density is low compared to the target density.

Molar Activity

The molar activity (Am) is the ratio of radioactivity to total mass of the tracer, expressed in Becquerel/mol. A sufficiently high Am of the PET tracer is a pre- requisite for administering doses that do not evoke a pharmacological or tox- icological response from the system under study. For low-density targets, often found in the central nervous system (CNS), a high Am is critical for visualization. Theoretical values are listed in Table 1 but in practice the values of Am achieved are much lower due to isotopic dilution processes during radionuclide production and synthesis of the tracer. In the case of carbon-11, the radionuclide utilized in papers I–II and IV–V, stable carbon isotopes can be introduced for example by carbonous contaminants in the target holder materials and in the target gas.63 The energy deposited by the proton beam into the nitrogen and oxygen gas used in carbon-11 production will readily transform the contaminants to carbon dioxide. Other sources could be impurities origi- nating from reagents and atmospheric carbon dioxide.

24 Physicochemical Properties Since many targets are located intracellularly or in the CNS, a certain degree of lipophilicity is needed to pass membranes by passive diffusion. However, excessively high lipophilicity, as measured by partition coefficients (logP or, preferably, logD), can cause problems with non-specific binding. The lipophilicity also affects binding to plasma proteins and thus the free fraction of the tracer in plasma.64 Attempts have been made to predict preferred physicochemical parameters for CNS tracers, taking into account the topological sur- face area, the molecular weight, pKa as well as number of hydrogen bond do- nors.65 Both the physicochemical properties of a tracer and its propensity to undergo active efflux are a direct result of its chemical structure.66–68 For a prospective CNS tracer, active transportation out of CNS will likely deem the candidate unsuitable as a PET tracer.

Metabolism The PET detector cannot distinguish between different sources of radiation, thus both signals from the parent tracer and any radioactive metabolites will be registered. Metabolism may or may not be an issue depending on the metabolites formed. Preferably, the tracer has high metabolic stability or the metabolites formed are not taken up in the target tissue; however (extensive) clearance of the tracer from blood must be corrected for. A metabolite of similar lipophilicity may lower the signal-to-noise ratio and a metabolite with similar affinity for the target will confound the quantification.69 The position of the label can therefore influence the impact of the inevitable metabolism.70

The complexity of PET tracer development thus becomes evident through the characteristics a successful tracer should possess. Many, if not all, of the parameters discussed are related to the structure of the tracer. Thus, starting from a set of structurally related compounds, all amenable for labeling, would be an advantageous starting point in a tracer development project.71,72 Further- more, reliable methods for the labeling should preferably already be at hand. From the preceding discussion, the early stages of PET tracer development could be envisioned as in Figure 5. The basic structure is largely dictated by the target but careful decoration of the structure will lead to a set of compounds, synthesized from a common precursor, which can be evaluated for affinity and selectivity for the target by in vitro techniques. Promising compounds can then be labeled and evaluated with ex vivo and in vivo studies. From the outcome of the initial pre-clinical studies, a choice can be made to investigate another compound or even to synthesize an entirely new chemical entity. This iterative workflow, aimed at finding a PET tracer, is greatly facilitated if the library of reference compounds can be synthesized from the same precursor as used in the labeling.

Figure 5. Workflow in early stages of PET tracer development.

11C-Carbonylative Radiochemistry Radiochemistry is limited to the number of primary and secondary labeled precursors available, which in turn limits the structures amenable for labeling and the reactions possible to perform. Further restraints are the half-lives of the radionuclides which prompt the labeling to be swift and toward the end of the synthetic route (Table 1). Carbon-11 is produced by the 14N(p,α)11C reac- 11 11 tion and primary precursors [ C]methane or [ C]carbon dioxide are formed via the addition of either hydrogen or oxygen gas to the nitrogen gas in the target.73 The half-life of carbon-11 is 20.4 min, considerably shorter than the 110 min of fluorine-18, but the main advantage of carbon-11 is the natural abundance of carbon atoms in organic molecules. The short half-life also allows for repeated PET scans in a study subject during a day but restricts the use to facilities with in-house cyclotrons. The first production of carbon-11 was performed in 1934, and the first biological experiments were performed with [11C]carbon dioxide in 1939, and [11C]carbon monoxide in 1945.74–77 Today, the majority of 11C-labeling reactions are performed with either [11C]methyl iodide or [11C]methyl triflate, to introduce a methyl group to a heteroatom.78–80 The simplicity of this labeling method has earned its widespread use and applications in palladium-mediated cross-coupling reactions have further broadened the scope.81 However, labeling with 11C-methylation reagents is limited to compounds with methyl groups 11 and the pendant [ C]CH3-group can be liable to metabolism. The work in this thesis has been focused on the incorporation of [11C]carbon monoxide as a part of PET tracer development (papers I and II) or method development (papers IV and V). The production of [11C]carbon monoxide from [11C]carbon dioxide has traditionally been performed online, with reduction over either zinc heated to 400 °C,82 charcoal heated to 900 °C83 or molybdenum heated to 850 °C,84 although recent methods have disclosed the use of silacarboxylic acids or solid supported zinc for the reduction.85–87 By use of transition metals, such as rhodium, selenium and palladium, or by thermal or photo initiation, [11C]carbon monoxide has been incorporated into a diverse array of 11C-labeled carbonyl derivatives, highlighting the structural

26 diversity attainable with 11C-carbonylation reactions (Figure 6).88–92 Further- more, the 11C-carbonylation reaction offers the possibility to assemble complex starting materials at the end of the synthetic route and the generally low natural abundance as well as the low reactivity of carbon monoxide offers the possibility to reach high levels of Am.

Ureas O R 11 C R' Sulfonyl carbamates N N Carbamates H H O O O O 11 11 S C R' 3 R C R'

N N O - R N O [R 2 2 R H

H h H

], , R ] -N

h R R S ,

O R'-NH ' R ] -O 2 - [ e OH H N [S 3 R'- O O [Pd], R-X 11 R-X Aldehydes 11 C [ C]CO 11 C R' Esters R H (Et)3SiH R'-OH, hv R O [P -X d R ], , ' [ ] R P O R

d R H - d - ' - X P -

[ M N ]

H R

2 O - X O Ketones 11 C Carboxylic acids R R' 11 C O R OH 11 C R' R N H Amides Figure 6. Selected 11C-labeled carbonyl-derivatives. Examples compiled from various sources.88,89,93–98

Despite its versatility, the use of [11C]carbon monoxide is limited to a few sites worldwide.99 The handling of the minute amounts of [11C]carbon monoxide produced, typically 10-100 nmol, and the limited solubility of carbon monoxide in many organic solvents together with the short half-life of carbon-11, pose restrictions on 11C-carbonylative chemistry.100,101 Thus, [11C]carbon monoxide needs to be confined in low-volume reaction vessels to achieve sufficiently high reactant concentrations in order to attain sufficiently fast reaction kinetics. Under such conditions, with reactants and reagents in large excess, the limiting factor for the reaction becomes the mass transfer of gas into the solution. Passing [11C]carbon monoxide through a reaction mixture does not lead to satisfactory conversion of [11C]carbon monoxide into 11C-labeled product, unless highly reactive reagents are present.83,98,102,103 Therefore, several conceptually different approaches have been investigated in order to circumvent this problem. An early approach adopted a re-circulation technique with [11C]carbon monoxide passing through the heated reaction mixture.90 The conversion of [11C]carbon monoxide to non-volatile products was increased, compared with

27 the single-pass method. A breakthrough came with the utilization of high pressure to concentrate [11C]carbon monoxide and the reagents into a small volume.104–106 The system, schematically depicted in Figure 7, is centered around a microautoclave. The process starts with the cyclotron production of [11C]carbon dioxide via the 14N(p,α)11C-reaction. [11C]Carbon dioxide is delivered to the hot cell in a stream of helium and concentrated on a solid support before online reduction to [11C]carbon monoxide over heated zinc granules. The [11C]carbon monoxide is concentrated before being transferred to the microautoclave. The reactants, already in solution, are held on an injection loop and transferred to the microautoclave under high pressure, thereby reducing the gas volume in the autoclave from 200 µL to less than 1 µL. The reaction with [11C]carbon monoxide is facilitated by the high pressure, as solubility in- creases with pressure. The reaction can be further facilitated by heating and the use of an autoclave permits temperatures above the boiling point of the solvent to be used. This experimental set-up has been utilized in the 11C-carbonylative synthesis of 11C-labeled amides,105 ketones,107 ureas108 and carboxylic acids,95 to name a few. The system was also employed for the 11C-aminocarbonylations presented in paper I.

CYCLOTRON

TARGET HOTCELL Zn oven + 14 11 11 11 p N(p, ) C [ C]CO2 [ C]CO 11 [ C]CO2 trap 11 [ C]CO2 trap

Waste Pump

Micro- Stop Waste Vent autoclave [11 C]CO trap

Product vial Oil bath

Injection loop Figure 7. Experimental set-up for the [11C]CO-system operating under high pressure.

Although successfully applied to the synthesis of various carbonyl-11C derivatives, the system depicted in Figure 7 is technically demanding and alternative approaches to confine the [11C]carbon monoxide by chemical means have been investigated. The first employed borane to complex [11C]carbon monoxide in solution,109 while later examples have utilized a copper(I) scorpionate complex103 or Xantphos as supporting ligand to trap [11C]carbon monoxide in the reaction mixture.85,86,98 The [11C]carbon monoxide is then released either by heating, the addition of a competing ligand or, in the case of Xantphos, by

28 continuing forward in the reaction. Further developments have adapted microfluidic reactors to the radiochemical setting.110–112 In 2012, Eriksson et al., disclosed the use of xenon as carrier gas.113 Xenon’s generally good solubility in organic solvents enabled the direct transfer of concentrated [11C]carbon monoxide into the reaction mixture without simultaneous venting of the carrier gas. Thus, the 11C-carbonylation reactions were possible to perform in a closed system at ambient pressure. The system is schematically depicted in Figure 8. The experimental set-up resembles the set-up in Figure 7, with the key feature of switching the carrier gas from helium to xenon to carry the concentrated [11C]carbon monoxide to the reaction vessel. The 11C-carbonylations described in papers II, IV and V were performed with this system.

Figure 8. Experimental set-up for the [11C]CO-system utilizing xenon as carrier gas.

Targeting the Cholinergic System The Cholinergic System Cholinergic neurons are widely distributed throughout the CNS.114,115 High densities of cholinergic neurons are found in the striatum, basal forebrain, spinal cord, medial habenula and olfactory tubercle while cholinergic innervation is found in virtually all regions of the brain. The neurotransmitter in the cholinergic system, acetylcholine (ACh), was the first neurotransmitter to be dis- covered in a chain of events stemming from the first synthesis in 1867 to the demonstration of parasympathetic neuronal transmission in a denervated frog heart in 1926.116–119 A schematic drawing of a cholinergic nerve terminal and the synthesis, storage and release of ACh is depicted in Figure 9.120

Figure 9. Schematic drawing of ACh synthesis, storage and release in the cholinergic synapse. Abbreviations: Acetylcholine (ACh), Acetylcholinesterase (AChE), Acetyl coenzyme A (Acetyl-CoA), Choline (Ch), High-affinity choline transferase (CHT1), Muscarinic receptor (M), Nicotinic receptor (N), Vesicular acetylcholine transporter (VAChT). Adapted from Prado et al.120

Choline is taken up into the cell via the high-affinity choline transporter (I). Choline acetyltransferase (ChAT) mediates the synthesis of ACh from acetyl coenzyme A and choline (II). The vesicular acetylcholine transporter (VAChT) loads ACh in synaptic vesicles (III). Upon stimulation of the nerve, the vesicles migrate and fuse with the cell membrane and release ACh out into the synapse (IV). The signal is conveyed via ACh binding to a cholinergic (nicotinic or muscarinic) receptor (V). The cycle is finally closed when ACh is degraded to choline and acetate by acetylcholinesterase (AChE) (VI).

The Cholinergic System in Neurodegenerative Diseases The cholinergic system is involved in modulation of several critical processes in the CNS,121–123 such as attention,124 arousal,125 memory126 and learning127. These cognitive functions are affected in various neurodegenerative diseases. The most notable example is Alzheimer’s disease (AD), a progressive neurodegenerative disease characterized by a rapid cognitive decline. This cognitive impairment has been correlated with a loss of cholinergic synapses where the specific depletion of cholinergic neurons in the basal forebrain has been linked to the memory loss seen in AD patients and the levels of cholinergic markers are lower in AD patients compared to normal controls.128–133 The association

30 between the cholinergic system and cognitive dysfunction led to the postula- tion of the “cholinergic hypothesis”.134 While this hypothesis has stimulated a large body of research over the years, recent discoveries have led to a more nuanced view on the disease mechanism.135 For example, studies of AD patients in early stages of the disease have found no decrease in ChAT activity and no measurable reduction of basal forebrain cholinergic neurons. Apart from AD, the cholinergic system is affected in a number of other neurodegenerative diseases.136 Patients with Parkinson’s disease, parkinsonian dementia, Huntington disease, dementia with Lewy bodies, Down syndrome, progressive supranuclear palsy and olivopontocerebellar atrophy also display alterations in the cholinergic system.137–144 Although the cholinergic degeneration is manifested as cognitive impairments, the pathology differs and different brain regions are affected in the various diseases.

There are biomarkers by which the integrity of the cholinergic system can be studied although most efforts have been directed toward understanding and assessing AD pathology.145–147 Several PET and single-photon emission computed tomography (SPECT) tracers have been developed for in vivo studies. The cholinergic receptors (nicotinic and muscarinic) are localized pre- and postsynaptically but PET tracers targeting either receptor will consequently only visualize a specific receptor and, possibly, only a specific subtype.148–150 AChE is found pre- and postsynaptically as well as intersynaptically and has been studied using PET with tracers targeting either AChE directly, such as [11C]physostigmine, or tracers acting as substrates for AChE, such as N- [11C]methylpiperidin-4-yl propionate ([11C]PMP).151,152 Although imaging AChE is advantageous for assessing the efficacy of AChE inhibitors in symp- tomatic treatment of AD, AChE has been found in non-cholinergic neurons, thus limiting its specificity as a cholinergic marker to some extent.153,154 VAChT is only found presynaptically and has been imaged with SPECT- tracer [123I]iodobenzovesamicol. The cholinergic neuronal density was determined in patients with multiple system atrophy, progressive supranuclear palsy, Parkinson’s disease and dementia with Lewy bodies.155–159 VAChT imaging has thus demonstrated the ability to assess the temporal and regional spatial changes in cholinergic neuronal density which could be of use for eval- uating neuroprotective treatments. However, substantial efforts have been directed toward the development of a VAChT PET tracer as SPECT cannot pro- vide as high sensitivity and spatial resolution as PET. Furthermore, PET allows for easier quantitative measurements and acquisition of dynamic images. The development of a PET tracer for VAChT is described in the next sections and in papers I and II.

31 The Vesicular Acetylcholine Transporter In the late 1960’s, a drug discovery program aimed at finding an analgesic drug resulted in the discovery of (±)-trans-2-(4-phenylpiperidino)cyclohexanol or vesamicol as the compound later became known.160,161 Vesamicol did not produce an analgesic effect but rather showed a peripheral neuromuscular effect.160 Further studies into the mechanism of action of vesamicol pointed towards a presynaptic effect on the storage of ACh in the nerve terminal.161– 164 Around the same time, nematodes with mutations on the unc-17 locus were affected in properties relating to cholinergic function.165–167 Further studies identified a protein with 40% amino acid similarity to the vesicular monoamine transporters and, with the aid of vesamicol, its identity as VAChT could be established.168–170 There are three families of proteins that mediate the uptake of neurotrans- mitters into synaptic vesicles and VAChT belongs to the same family as the two vesicular monoamine transporters 1 and 2.171 In the human genome, the VAChT gene is found entirely within the first intron of the ChAT gene, an arrangement called “the cholinergic locus”.168 VAChT has been shown to be a crucial component of the cholinergic system. Homozygous VAChT knock- out mice died shortly after birth due to asphyxiation.172 The regulation of the cholinergic synapse during fetal development was found to be reliant on VAChT as well. Selective deletion of VAChT in mouse brain caused deficits in learning and spatial memory in addition to hyperactivity.173–175 Based on the protein sequence, VAChT has been predicted to have 12 transmembrane domains which fold into two bundles, each containing six heli- ces.176 The transportation of ACh by VAChT is closely coupled to an H+- ATPase.177 ACh is transported into the vesicle by exchange of two protons from the vesicle lumen. The distribution of VAChT in CNS has been visualized with immunohisto- chemical methods.178–181 In rat, monkey and human brain tissues, VAChT was found in the cerebral cortex, basal forebrain, hippocampus, several thalamic nuclei, hypothalamus, amygdala and striatum, all areas known to contain cholinergic innervation. Moreover, the ChAT and VAChT immunoreactivity overlapped thus confirming VAChT as a biomarker for cholinergic neurons.180–182

Development of Ligands for the Vesicular Acetylcholine Transporter

Vesamicol is an allosteric inhibitor of VAChT, with measured Ki values ranging from 2.0 nM to 47 nM depending on assay design (Figure 10).162,164,183–188 The binding of vesamicol to VAChT is stereoselective, with (R,R)-vesamicol showing a higher affinity compared to distomer (S,S)-vesamicol. However, the exact binding site on VAChT is unknown.164,185,186 Vesamicol has served

32 as a single lead compound despite its affinity to σ receptors and α-adrenocep- tors apart from VAChT.189–191 The affinity for the σ receptors in particular has been a challenge in the design of new VAChT ligands. In 2012, an insecticide based on a spiroindoline structure was found to target VAChT and the scaffold was investigated as a new lead.192,193 However, the affinities of the spiroindoline derivatives toward VAChT were only low to moderate, and their affinities for the σ receptors were in the same range. Thus, there was little or no selectivity for VAChT.

Figure 10. Model compounds for the different categories of VAChT ligands.

The VAChT ligands synthesized thus far can roughly be divided into different categories based on their structure: vesamicol-based, benzovesamicol-based and bipiperidyl-based (Figure 10).183,185,189,194 Based on the findings disclosed so far, the following can be deduced about the structure-activity relationship of an aspiring VAChT PET tracer:

 The VAChT binding site is enantioselective and the (-)-enantiomer has often shown a higher affinity for VAChT than the (+)-enantiomer.185,186,195–199  A trans relationship between the hydroxyl group and the amine is beneficial.185  The hydroxyl group is essential for affinity, being either absent or esterified causes a loss in affinity.185  A rotationally constrained scaffold is preferable, as open-chain ligands tend to have low affinities for VAChT.184,185,194,200

These structural necessities are found in the majority of VAChT ligands synthesized in the last three decades. Giboureau et al. published two extensive reviews in 2010; one of which focused on the development of VAChT ligands.189 Numerous ligands based on either structural category have been synthesized and many of them have shown low nanomolar affinity for VAChT,

33 184,186,188,195,197–199,201–203 defined here as a Ki < 10 nM. This strongly suggests the vesamicol scaffold to be a valid lead, though poor selectivity for VAChT over the σ receptors rendered many potent ligands unsuitable for further evaluation.186,188,201 This selectivity issue is caused by an overlapping pharmacophore for the VAChT binding site and the σ receptor binding site, which is not limited to vesamicol-derivatives as demonstrated by the spiroindoline insecticide which was found to bind to the same site as vesamicol.183,193,204,205 The second review by Giboureau et al. focused on the preclinical evalua- tions of 11C/18F-labeled VAChT ligands.206 Only a few have progressed to studies on non-human primates (NHP) or human subjects and fewer still are regarded as viable candidates for PET imaging of VAChT.203,207–212 The rea- sons for failure are varied but poor in vivo selectivity, rapid metabolism and low uptake in the brain were seen in rat and NHP PET studies. VAChT ligands evaluated in human subjects, (–)-5-[18F]fluoroethoxybenzovesamicol ([18F]FEOBV), (+)-4-[18F]fluorobenzyltrozamicol ([18F]FBT) and (–)-N- ethyl-N-[18F]fluoroacetamidobenzovesamicol ([18F]NEFA, are shown in Fig- ure 11.

Figure 11. VAChT ligands evaluated in PET studies of NHP or human subjects.

Here, [18F]FBT showed poor in vivo selectivity for VAChT over the σ receptors whereas [18F]NEFA was poorly retained in the cortex of controls and AD subjects.206,213 [18F]FEOBV has shown an ability to detect differences in brain

34 cholinergic losses in lesioned rats and was recently approved for studies on human subjects.214–216 The brain biodistribution was consistent with cholinergic nerve terminals but the time to reach steady state was more than 360 min post injection. These slow equilibrium kinetics were also seen in NHP and can be problematic when imaging patients with cognitive deficits.208 Concerns have also been raised regarding the in vivo selectivity of [18F]FEOBV.183,217 As an alternative to [18F]FEOBV, the synthesis of benzovesamicol-based VAChT ligands with a carbonyl-group inserted between the B- and C-ring has recently rendered several potent and selective ligands.195,197,199,201 Encouraging results have been demonstrated by (–)-[11C]TZ659 and (–)-[18F]VAT in NHP studies but this subclass is yet to be evaluated in human subjects (Figure 11).198,218–220 In this thesis, the design, synthesis, 11C-labeling and preclinical evaluation of benzovesamicol-based VAChT ligands is presented in papers I and II.

35 Design, Synthesis and 11C-Labeling of Ligands for the Development of a PET Tracer for VAChT

The intricacy of the cholinergic system is evidenced in a way by the number of pathologies in which it is affected. Thus, studying the cholinergic system would offer insights into the etiology and treatment of these pathologies. Here, non-invasive in vivo imaging studies using PET presents the option to quantify both temporal and spatial changes of the cholinergic innervation and to evaluate therapeutic outcomes. With a high affinity ligand (vesamicol) on hand, a lot of effort has been directed toward the development of a PET tracer for the cholinergic marker VAChT but of the numerous ligands synthesized thus far no candidate has found routine clinical use.189,206 The main reason for failure has been a lack of affinity and/or poor selectivity for VAChT over the σ receptors as determined by in vitro experiments. Despite this, elevated potency and selectivity have not been a guarantee for success of the VAChT ligand in ex vivo and in vivo studies. Pitfalls related to the affinity, specificity and selectivity as well as the kinetic properties have rendered many ligands unsuitable. How can this be addressed? The importance of the chemical structure can be inferred from the discussion in the section Considerations in Tracer De- velopment. Although this may seem obvious, it should be recognized that structural changes may have an impact not only on affinity and selectivity, but on kinetic properties as well.221 Thus, careful consideration of which structural components to incorporate apart from the pharmacophore dictated by the target, is needed as the structure should present opportunities for labeling as well as late-stage modifications to ease the transition between ligand design and experimental results as shown in Figure 5.

36 Synthesis and Labelling of a Piperazine-Based Library of 11C-Labeled Ligands for Imaging of the Vesicular Acetylcholine Transporter (Paper I)

Aim The main objective of paper I was to find a promising PET tracer candidate for VAChT through the design and synthesis of a set of piperazine-based ligands and their subsequent 11C-labeling and preclinical evaluation (Figure 12). Of particular importance was the development of a generic 11C-aminocarbonylation method, for 11C-labeling of all ligands.

Figure 12. Workflow in paper I.

Design, Synthesis and 11C-Labeling The scaffold was influenced by the work of Tu et al. who, in repeated studies, have incorporated a carbonyl group between the B and C-ring of benzovesamicol.195,197,201 Several of the carbonyl-containing derivatives displayed high affinities for VAChT in the low nanomolar region. Here, the carbonyl group offered a position for incorporation of [11C]carbon monoxide and by changing the piperidine ring (B) for a piperazine ring, a versatile 11C-aminocarbonylation protocol could be developed. Through variation of the electrophilic substrate, a set of structurally related ligands would be possible to 11C-label and evaluate preclinically. Incorporation of a piperazine ring has previously been explored and the results have been encouraging in the form of high affinity for VAChT and demonstrated brain uptake.188,209,222,223 Synthesis of ligands (±)4a–f, to be used as references, started from 1,4- dihydronapthalene and is outlined in Scheme 1. Reaction with meta-chloroper- oxybenzoic acid (mCPBA) furnished epoxide 1.224 Opening of the epoxide with benzyl piperazine-1-carboxylate (1-Cbz-piperazine) gave (±)2, which was deprotected by catalytic dehydrogenation to (±)3.225,226 Ligands (±)4a–f were synthesized via a peptide coupling with the corresponding aryl carboxylic acid.227

Scheme 1. Synthesis of reference ligands (±)4a–f. I) mCPBA, CHCl3, 0 ºC to rt, 20 h, 79% II) 1-Cbz-piperazine, LiClO4, CH3CN, reflux, 22 h, 66% III) Pd/C, H2, EtOH, CH2Cl2, 10 bar, rt, 5 h, 97% IV) Aryl carboxylic acid, HBTU, DIPEA, CH2Cl2, rt, 1–22 h, 64%–77%.

For synthesis of the 11C-labeled analogs, a palladium-mediated 11C-aminocarbonylation of (±)-3 with the corresponding aryl iodide furnished 11C-(±)4a–f (Table 2). The RCY was based on the amount of [11C]carbon dioxide delivered to the hot cell and the activity of the 11C-labeled product isolated after semi- preparative HPLC (decay-corrected). Amides 11C-(±)4a–c and 11C-(±)4e were isolated with lower radiochemical yields (RCY, 4%–10%) compared with 11C-(±)4d and 11C-(±)4f (22% and 25%). The conversion of [11C]carbon monoxide to non-volatile 11C-labeled products was in the same range except for 11C-(±)4a. The results were satisfactory given that no optimization for indi- vidual reactions had been performed. In light of this, the values of Am obtained for 11C-(±)4a–f were also encouragingly high (124 GBq/µmol–597 11 GBq/µmol). The Am was determined from the activity of the C-labeled amide at the end of synthesis following a large irradiation (> 15 µAh).

38 Table 2. 11C-aminocarbonylation for synthesis of 11C-(±)4a–f

11 11 a C-labeled [ C]CO-conversion b c d RCY (%) Am (GBq/µmol) RCP (%) ligand (%) 11C-(±)4a 42 ± 10 (2) 10 ± 1 (2) 164 ± 46 (2) 95 ± 4 (2) 11C-(±)4b 65 ± 4 (2) 10 ± 3 (3) 337 ± 87 (3) 97 ± 2 (3) 11C-(±)4c 68 ± 3 (3) 4 ± 3 (2) 203 ± 20 (2) 96 ± 2 (2) 11C-(±)4d 65 ± 9 (2) 25 ± 5 (3) 255 ± 130 (3) 97 ± 1 (3) 11C-(±)4e 70 ± 2 (2) 9 ± 3 (3) 124 ± 79 (3) 96 ± 2 (3) 11C-(±)4f 66 ± 5 (5) 22 ± 7 (2) 597 ± 208 (2) > 99 (2) Reaction conditions: (±)3 (13 µmol), aryl iodide (39–50 µmol), Pd(PPh3)4 (1.7 µmol), THF (600 µL). Number of experiments in brackets. aFraction of [11C]CO converted to non-volatile compounds. Decay-corrected. bBased on the radioactivity of the isolated 11C-labeled ligand at 11 c EOS and the amount of [ C]CO2 delivered to the hot cell. Decay-corrected. Based on the radioactivity of the isolated 11C-labeled ligand at EOS and the concentration determined by analytical HPLC. dDetermined by HPLC analysis of isolated 11C-labeled ligand.

Preclinical evaluation Assessment of 11C-(±)4a–f binding to VAChT and σ receptors was performed by autoradiography studies using mouse and rat brain tissue along with human postmortem brain tissue from AD patients.71,72 Vesamicol and 1,3-di-o-tolylguanidine (DTG) were used to block the specific binding to VAChT and the σ receptors, respectively.228 Of 11C-(±)4a–c, only 11C-(±)4b showed accumulation in the brain tissues, however an accumulation which proved to be non- displaceable (not shown). This lack of accumulation was surprising, as the piperidine analogs of (±)4a–c had displayed binding to VAChT in in vitro 195 binding studies (Ki-values < 3 nM). Of 11C-(±)4d–f, only naphthyl-derivatives 11C-(±)4d and 11C-(±)4e displayed specific binding and the binding in all tissues could be displaced, albeit to a lesser extent when using the reference compound (Figure 13). Their lip- ophilic character would make them prone to bind in an unspecific fashion to the brain white matter (cLogD7.4 3.3–3.4).

Figure 13. Autoradiograms of 11C-(±)4d (left) and 11C-(±)4e (right). Depicted is the total and non-specific binding in tissue from rat, mouse and human AD brains. Adapted from paper I.

To learn more about this piperazine-based scaffold, small animal PET studies on 11C-(±)4b and 11C-(±)4d–e were conducted (Appendix 1). Here, 11C-(±)4b showed the highest rat brain uptake with a maximum standardized uptake value (SUV) of 4.5 shortly after injection followed by a rapid wash-out (Fig- ure 14, left). For 11C-(±)4d–e, the brain uptake was found to be negligible. Organ distribution studies found all three 11C-labeled ligands to accumulate in the liver, intestine and kidneys (Figure 14, right).

Figure 14. Small animal PET studies of 11C-(±)4b, 11C-(±)4d–e, showing (left) SUV time-activity curves of rat brain uptake and (right) organ distribution data.

The results from the preclinical experiments are conclusive on the unsuitability of 11C-(±)4a–f as PET tracers for VAChT. Ligands 11C-(±)4b and 11C- (±)4d–e all showed binding in the brain tissue samples, although the extent of specific binding varied. The lack of brain retention in the small animal studies

40 further suggested the binding to VACHT to be low although 11C-(±)4b showed some initial retention. It should be noted that an immediate, extensive metabolism of 11C-(±)4b, and 11C-(±)4d–e, would affect the in vivo brain accumulation but metabolism cannot explain the autoradiographic findings. It was surprising that the exchange of a carbon atom for a nitrogen atom would have such an impact on the binding to VAChT that it had for 11C-(±)4a– c and their piperidine analogs but this might be related to their conformation. Crystal structures of VAChT ligands, including the piperidine analog of (±)4a, all show a perpendicular arrangement of the B- and C-ring.185,194,195 With piperidine exchanged for piperazine, a planar conformation would allow orbital overlap between the aryl and the amide thus hindering free rotation around the amide single bonds. A perpendicular arrangement cannot be reached and, if this also is the active binding conformation to VAChT, the lack of binding displayed by 11C-(±)4a, 11C-(±)4c and 11C-(±)4f can be explained. An addi- tional steric effect, forcing the naphthyl rings out of the amide plane may account for the binding of 11C-(±)4d and 11C-(±)4e.

Synthesis and In Vitro Evaluation of 5-Substituted Benzovesamicol Analogs containing N-Substituted Amides as Potential Positron Emission Tomography Tracers for the Vesicular Acetylcholine Transporter (Paper II)

Aim The main objective in paper II was to continue the search for a VAChT PET tracer candidate, this time via the design and synthesis of a set of ligands and in vitro evaluation of their affinity and selectivity for VAChT (Figure 15). As in paper I, inclusion of an amide functional group would allow both a facile synthesis of structurally related ligands for structure-activity determinations and 11C-labeling through an aminocarbonylation reaction using [11C]carbon monoxide.

Figure 15. Workflow in paper II.

Design and Synthesis of 5-Substituted Benzovesamicol Analogs containing N-Substituted Amides For paper II, the idea was to explore substitution on C5 of the tetrahydronaphthol ring. This position has previously been successfully functionalized, in addition to the other positions on the aromatic ring.183,185,198,229,230 Derivatization at C5 has started from 1-aminonaphthalene to give 5-aminobenzovesamicol (ABV), a high affinity VAChT ligand.183,185,201,230,231 ABV has then been further functionalized in order to reach the final VAChT ligands. In order to be able to explore both aromatic and aliphatic substituents at C5, variation of the amine component was preferred. An electrophilic precursor was therefore sought, one that could be utilized both for synthesis of the ligands and for their 11C-labeling. Utilizing α-naphthol as starting material, a triflate precursor was identified as a viable alternative that could be synthesized via a shortened synthetic route compared to ABV (Scheme 2).

Scheme 2. Synthesis of precursor (±)8. I) NH3 (l), Li, EtOH, -78 °C, 1 h, 73% II) N-Phenyl- bis(trifluoromethanesulfonimide), K2CO3, THF, MW, 120 °C, 6 min, 62% III) mCPBA, CH2Cl2, 0 °C to rt, 16 h, 77% IV) 4-Phenylpiperidine, LiClO4, CH3CN, reflux, 16 h, 33% ((±)8) and 49% ((±)9).

A Birch reduction of α-naphthol furnished 5,8-dihydronaphthol 5, which was converted to triflate 6 by a microwave-assisted protocol.232,233 mCPBA was used again for epoxidation, here of the isolated double bond in 6, to give race-

42 mate (±)7.224 An over-night epoxide opening with 4-phenylpiperidine followed by a tedious separation gave regioisomers (±)8 and (±)9, identified by 2D-NMR.225 An aminocarbonylation protocol, developed in-house, was used for the synthesis of ligands (±)10a–n (Scheme 3).234 Apart from the previously C5- functionalized VAChT ligands mentioned, 3D quantitative structure-activity relationship (QSAR) studies had found that steric bulk could be tolerated on the benzovesamicol ring, preferably in the C5-position.202,235 Different amines were therefore selected to explore more comprehensively how substituents on the amide nitrogen would affect the binding affinity to VAChT and the selectivity over the σ receptors.

Scheme 3. Synthesis of ligands (±)10a–n. I) Amine, Herrmann’s palladacycle, XPhos, Mo(CO)6, DMAP, Cs2CO3, 1,4-dioxane or CH3CN, 160 °C, MW, 3 h–8 h, 5%–37% yield (isolated as TFA-salts).

In Vitro Evaluation

The binding of ligands (±)10a–n to VAChT and the σ receptors was assessed by an in vitro binding assay (Table 3). The binding affinities for VAChT ranged from micromolar to low nanomolar levels for the evaluated ligands.

43 Table 3. Inhibition constants (Ki) of (±)benzovesamicol, (±)10a–n, (+)10g, (–)10g and (±)11

Ligand Ki ± SD (nM) Selectivity factor

VAChT σ1 σ2 Ki (σ1) / Ki (σ2) / Ki (VAChT) Ki (VAChT) (±)benzo- 1.3 ± 0.5 119 ± 6 96.4 ± 22.3 92 74 vesamicola (±)10a 2107 ± 791 4281 ± 575 808 ± 78 2.0 0.4 (±)10b 950 ± 4 874 ± 289 368 ± 13 0.9 0.4 (±)10c 295 ± 47 > 10 000 9950 ± 7142 > 34 34 (±)10d 2900 ± 849 1900 ± 283 > 10 000 0.7 > 3.4 (±)10e > 10 000 > 100 000 > 30 000 - - (±)10f 205 ± 11 1225 ± 792 475 ± 54 5.9 2.3 (±)10g 67.0 ± 25.4 1326 ± 142 384 ± 36 20 5.7 (+)10g 393 ± 69.6 6214 ± 2144 915 ± 81 16 2.5 (–)10g 56.7 ± 20.8 2974 ± 1242 612 ± 270 52 11 (±)10h 196 ± 26 1968 ± 83 498 ± 96 10 2.5 (±)10i 147 ± 2 613 ± 133 278 ± 40 4.2 1.9 (±)10j 63.5 ± 14.1 1662 ± 236 468 ± 71 26 7.4 (±)10k 582 ± 159 6937 ± 2010 7190 ± 1050 12 12 (±)10l 206 ± 18 1902 ± 83 1607 ± 513 9.2 7.8 (±)10m 249 ± 11 725 ± 156 1718 ± 747 2.9 6.9 (±)10n 341 ± 33 > 10 000 1359 ± 80 > 29 4.0 (±)11 795 ± 56 241 ± 37 2387 ± 14 0.3 3.0 3 3 Radiolabeled ligands ([ H]vesamicol for VAChT, (+)-[ H]pentazocine for σ1 receptor and 3 [ H]DTG for σ2 receptor) were used with (±)10a–n, (+)10g, (–)10g or (±)11 for the competitive a binding studies. The Ki-values are expressed as the mean ± SD (nM) for 2–7 experiments. Re- ported results are from Barthel et al.183

Ligands (±)10a–b and (±)10d–e with large, aliphatic N-substituents displayed the lowest affinities for VAChT. Ligands (±)10c and (±)10f with smaller, yet aliphatic, N-substituents had affinities similar to ligands (±)10g–n with a (hetero)aromatic N-substituent. In general, the affinities of ligands (±)10g–n were moderate to good, with benzyl derivatives (±)10g and (±)10j displaying the highest affinities for VAChT in the series (67 nM and 64 nM, respectively). The methylated analog (±)10h showed a loss in affinity compared to (±)10g, suggesting the N-H fragment of the amide either to be involved in binding to VAChT or to impose a favorable conformation. No electronic effect on the affinity could be discerned for (±)10i–m, whereas a slightly higher affinity was seen for benzyl derivatives (±)10g–j compared to aniline derivatives (±)10l–m. It is possible that the one-carbon chain shifts the aromatic ring to a favorable position, which cannot be reached by a zero- or two-carbon chain. The affinity of (±)10k contradicts this theory but the discrepancy can be because of the smaller size and more polar character of the thiazole-ring compared to the benzene-ring.

44 Prompted by the stereoselective binding properties of VAChT, racemate (±)10g was separated to (+)10g and (–)10g by supercritical fluid chromatog- raphy (SFC).164,236,237 While only a modest gain in affinity was reached, euto- mer (–)10g showed seven times higher affinity for VAChT than (+)10g (57 nM and 393 nM, respectively). The (–)-enantiomer displaying the higher affinity is in line with previously reported literature.186,195,197 A regioselective preference for C5-substitution over C8-substitution has also been reported and to investigate whether this was the case for the scaffold presented herein, (±)11 was synthesized from (±)9 and benzylamine.183,185,198,201,230 The affinity for VAChT was 12 times lower than for the C5-regioisomer (+)10g (795 nM and 67.0 nM, respectively). The ligands tended to show a higher selectivity for VAChT over the σ1 receptor irrespective of an aromatic or aliphatic sidechain. Interestingly, benzovesamicol-derived VAChT ligands modified in the C-ring have shown a 183,195,197,201 higher selectivity for VAChT over the σ2 receptor. Thus, the modifications in the A-ring pursued here can be a strategy for improving the selectivity over the σ1 receptor. The ligands with poor affinity for VAChT showed affinities in the same range for the σ receptors. The selectivity for VAChT over either σ receptor was therefore close to or even below 1 for ligands (±)10a–b and (±)10d–e. This was not the case for the more potent ligands, where the results showed independent variation in the affinities for VAChT and the σ receptors. The gain in affinity for VAChT shown by (–)10g compared to the racemate, was accompanied by a loss in affinity for both σ receptors. Similarly, although the affinities for all three targets decreased, the selectivity factors for (±)10h were not improved compared to (±)10g. When summarizing the results from the binding studies for this set of benzovesamicol-based ligands, with an N-substituted amide in the C5-position, it could be seen that the size of the nitrogen substituent was a more important determinant for VAChT affinity than electronic effects. This was also true for the selectivity factor. The best ligands, (±)10g, (–)10g and (±)10j, displayed Ki-values between 57 nM and 67 nM though even lower values would have been desirable for continued preclinical evaluation.183,238,239 The structures are nevertheless of interest for future optimization.

11C-Aminocarbonylation With the selected scaffold, an 11C-aminocarbonylation of triflate (±)8 would be possible for 11C-labeling of the desired ligands. Triflates have previously been explored as electrophilic substrates in 11C-carbonylations, in the synthesis of 11C-labeled amides and 11C-labeled ketones.107,240–242 The oxidative addition of an aryl triflate to a metal center is slower compared to an aryl iodide and an additive had therefore been used in the 11C-carbonylations of aryl triflates.243–245 Lithium bromide and tetrabutylammonium iodide (TBAI) were both tested here, but only TBAI gave product. Different palladium ligands

45 were tested in order to find a suitable ligand for the 11C-aminocarbonylation of (±)8, set up in the xenon system (Table 4). Although the [11C]CO-conversion was above 54% for all ligands tested, only Xantphos (entry 2) and Xphos (entry 6) gave the desired product. With Xantphos, the [11C]CO-conversion was almost quantitative but the amount of 11C-(±)10a formed was very low, giving an estimated RCY of 4%. The product selectivity for Xphos was higher, returning 11C-(±)10a in 11% RCY although the [11C]CO-conversion was among the lowest for the ligands tested.

Table 4. Selection of ligand for 11C-aminocarbonylation

Entry Ligand [11C]CO- Product RCYc (%) conversiona (%) selectivityb (%) 1 PPh3 67 ± 11 No product - (2) 2 Xantphos 97 ± 0 5 ± 2 4 ± 1 (2) 3 Dppf 83 No product - (1) 4 P(o-tol)3 54 No product - (1) 5 Dppp 54 No product - (1) 6 Xphos 55 ± 19 21 ± 2 11 ± 3 (3) Reaction conditions: (±)8 (2.2 µmol), dipropylamine (51 µmol), Pd(dba)2 (2.6 µmol), 2 or 4 a equivalents of ligand (in relation to Pd(dba)2), TBAI (2.7 µmol), THF (200 µL). Percentage of [11C]CO converted into non-volatile products. Decay-corrected. bPercentage of product formed, based on analytical HPLC of crude reaction mixture. cRadiochemical yield, estimated from the [11C]CO-conversion and product selectivity. Number of experiments in brackets.

To fully test the reaction, 11C-(±)10g, one of the best ligands in the series, was synthesized from benzylamine and (±)8 and isolated in 9% RCY and > 99% RCP (Scheme 4). The RCY was based on the starting activity of [11C]carbon monoxide transferred to the reaction vial and the activity of 11C-(±)10g isolated after semi-preparative HPLC (decay-corrected). Because of precipitation in the reaction vial, the reaction conditions from Table 4 had to be altered slightly, to enable the purification. The molar activity of 11C-(±)10g was determined at the end of synthesis in two experiments. Starting from 16.4 GBq of [11C]carbon monoxide, 11C-

46 (±)10g was isolated with 411 MBq giving an Am of 55 GBq/µmol. In the sec- 11 ond experiment, C-(±)10g was isolated with 235 MBq and an Am of 78 GBq/µmol, starting with 10.1 GBq of [11C]carbon monoxide.

11 11 Scheme 4. Synthesis of C-(±)10g. I) Benzylamine, Pd(dba)2, Xphos, [ C]CO, NEt3, TBAI, THF, 150 °C, 5 min, 9 ± 0.3% RCY (average of two experiments, decay-corrected).

Summary of Papers I and II The early stages of PET tracer development revolves around the search for a chemical entity with the right affinity and selectivity for the desired target and with structural features making it amenable for radiolabeling with an appro- priate PET-active radionuclide. The work presented in papers I and II has been concerned with the development of a PET tracer candidate for VAChT. Two different scaffolds have been explored as well as two different methods for assessing the affinity to VAChT. However, the main strategy has been the same, that the same precursor should be utilized for the synthesis of structurally related ligands and their subsequent labeling. This approach was time-efficient as there was no need to develop unique synthetic routes for different precursors to be used in the synthesis of ligands or in the labeling reactions. Paper I investigated a piperazine-based benzovesamicol scaffold and six ligands were labeled by an 11C-aminocarbonylation. Their affinity for VAChT was assessed by autoradiography studies with only 11C-(±)4b and 11C-(±)4d– e showing accumulation in brain tissue, albeit displaceable only to a certain extent. The small animal PET studies further corroborated their unsuitability. In paper II, substitution around the C5-position on benzovesamicol was examined by synthesis of fifteen N-substituted amide derivatives by an aminocarbonylation followed by their evaluation in an in vitro binding assay. The 11 best ligands had Ki-values below 67 nM and the possibility of C-labeling was demonstrated by the 11C-aminocarbonylation of 11C-(±)10g.

47 Although no ligand was found amenable for further preclinical evaluation, there are findings worth highlighting for future PET tracer development both in general and for VAChT specifically. The generality of the 11C-aminocarbonylation protocol developed in paper I and the ready binding assessments of ligands 11C-(±)4a–f was consistent with the strategy set out in the beginning of this section. The low-nanomolar affinity to VAChT and the selectivity over the σ1-receptor displayed by (–)10g in particular, from paper II, together with the demonstrated 11C-labeling of (±)10g, make the N-benzyl amide benzovesamicol ligands interesting for further modifications.

48 Method Development: Palladium-Mediated Incorporation of [11C]Carbon Monoxide or Carbon Monoxide

The ability of palladium to create new C-C or (C-heteroatom bonds) has earned its recognition because of the importance of being able to form C-C bonds under comparatively mild conditions and with tolerance to diverse functional groups. In the carbonylation reaction, one or two new C-C bonds are formed depending on the nucleophile. Furthermore, the abundance of carbonyl containing functional groups in biologically active compounds still makes the (Heck) carbonylation reaction worth exploring despite the advance- ments in the field since it’s pioneering almost fifty years ago. While the preceding section dealt with PET tracer development, where 11C/12C-carbonylation reactions were utilized as tools to synthesize and enable evaluation of the ligands, the present section will deal with the development of methods for incorporation of [11C]carbon monoxide or carbon monoxide by expanding on the advantages with the carbonylation reaction. Namely, the ability to utilize various, simple starting materials to reach more complex products and the ability to vary the carbon isotope incorporated according to the application.

Palladium and Visible-Light Mediated Carbonylative Suzuki-Miyaura Coupling of Unactivated Alkyl Halides and Aryl Boronic Acids (Paper III) Alkyl halides have been elusive substrates in transition metal-mediated reactions because of their slow oxidative addition and the risk of beta elimination as discussed in the section on The Palladium Mediated Carbonylation Re- action.26,28,29 During the last decade, the use of alkyl halides as cross-coupling partners has increased with the application of radical chemistry to the traditional cross- coupling protocols.246 By creating a single-electron transfer (SET) event, in which an alkyl radical is generated, the challenges related to the oxidative addition step can be circumvented. The alkyl radical can be generated in catalytic

49 amounts with the aid of an organometallic- or organic dye-based photocatalyst. Upon irradiation with visible light, the photocatalyst forms an excited state species capable of transferring an electron to generate the alkyl radical. By employing visible-light photocatalysis, unactivated alkyl halides have been used as substrates in cross-coupling reactions and functionalized under mild conditions and displayed great functional group tolerance.246 For carbonylative cross-couplings with alkyl halides as substrates, SET methodology has been employed but the generation of the alkyl radical has varied. Notably, whereas visible-light photocatalysis has been utilized in an aminocarbonylation,247 the methods have generally applied a combination of intense light or UV irradiation, elevated carbon monoxide pressures or elevated temperatures.248–257

Aim The main objective of the study was to develop a method that allowed access to unactivated alkyl halides as substrates in a carbonylative Suzuki-Miyaura coupling. Aryl alkyl ketones are found in biologically active compounds, such as VAChT ligands (Figure 11) and haloperidol-derived neuroleptics.195,258 In addition to the development of a methodology that would allow access to this substrate class, it was imperative that the method would offer mild reaction conditions, such as low-energy light irradiation and operate at ambient temperature and carbon monoxide pressure, to overcome the drawbacks of related methods (Figure 16).248,249,253

Figure 16. Schematic reaction set-up for the carbonylative Suzuki-Miyaura coupling.

Optimization of Reaction Conditions Investigation of the reaction conditions began using an iridium photocatalyst, 2 tris[2-phenylpyridinato-C ,N]iridium(III) (fac-Ir(ppy)3), and a palladium cross-coupling catalyst, bis(triphenylphosphine)palladium(II) dichloride (Pd(PPh3)2Cl2), to synthesize aryl alkyl ketone 14a from cyclohexyl iodide 12 and phenyl boronic acid 13 (Table 5).253,259

50 Table 5. Investigation of reaction conditions for carbonylative Suzuki-Miyaura coupling

a Entry Solvent Crxn Catalyst HE (eq) Temp NMR yield CCO 1 Benzene/H2O (2:1) Pd(PPh3)2Cl2, fac-Ir(ppy)3 2 rt 17% 2 Benzene Pd(PPh3)2Cl2, fac-Ir(ppy)3 2 rt 7% + 100 µL H2O 3 Benzene Pd(PPh3)2Cl2, fac-Ir(ppy)3 2 rt - 4 Acetonitrile/ Pd(PPh3)2Cl2, fac-Ir(ppy)3 2 rt - H2O (2:1) 5 Benzene/H2O (2:1) Pd(PPh3)4, fac-Ir(ppy)3 2 rt 24% 6 Benzene/H2O (2:1) Pd(dppf)Cl2, fac-Ir(ppy)3 2 rt 7% 7 Benzene/H2O (2:1) Pd(dba)2, XantPhos 2 rt - fac-Ir(ppy)3 8 Benzene/H2O (2:1) Pd(dba)2, SPhos 2 rt Trace fac-Ir(ppy)3 9 Benzene/H2O (2:1) Pd(PPh3)4, fac-Ir(ppy)3 2 70 °C 31% 10 Benzene/H2O (2:1) Pd(PPh3)4, fac-Ir(ppy)3 2 70 °C 31% 11 Benzene/H2O (2:1) Pd(PPh3)4, fac-Ir(ppy)3 1 70 °C 31% 12 Benzene/H2O (2:1) Pd(PPh3)4, fac-Ir(ppy)3 0.5 70 °C 46% 13 Benzene/H2O (2:1) Pd(PPh3)4, fac-Ir(ppy)3 0.25 70 °C 35% 14 Dioxane/H2O (2:1) Pd(PPh3)4, fac-Ir(ppy)3 0.5 70 °C Trace 15 Benzene/H2O (2:1) Pd(PPh3)4 - 70 °C 49% 16 Benzene/H2O (2:1) Pd(PPh3)4 - 70 °C 64% 17 Benzene/H2O (2:1) Pd(PPh3)4 - 70 °C 71% (65%) 18 Toluene/H2O (2:1) Pd(PPh3)4 - 70 °C 70% 19 Anisole/H2O (2:1) Pd(PPh3)4 - 70 °C (23%) Reaction conditions: Chamberrxn: 12 (0.25 mmol, entries 1-16; 0.30 mmol, entries 17-19), 13 (1.5 equiv), [Pd] (5 mol%), Xantphos (5 mol%), SPhos (10 mol%), fac-Ir(ppy)3 (1 mol%, entries 1-9; 2 mol%, entries 10-14), Hantzsch ester, K2CO3 (1 equiv), solvent (2 mL, entries 1-15; 4 mL, entries 16-19). ChamberCO: Mo(CO)6 (2.5 equiv), DBU (5 equiv), CH3CN (2 mL, entries 1-15; 4 mL, entries 16-19). aDetermined by 1H NMR, using anisole as internal standard. Isolated yield in brackets.

Using a CO-surrogate, Mo(CO)6, in a double-chamber system under blue and white LED irradiation, 14a was obtained in 17% NMR yield (entry 1).260,261 Having a two-solvent system rather than using H2O as an additive was important, as addition of only 100 µL of H2O resulted in a yield of 7% (entry 2) and using only benzene abolished product formation (entry 3).253 When altering the palladium catalyst (entries 5-8), only tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) improved the yield (24%, entry 5). Heating the CO- releasing chamber to 70 °C also led to an improvement (31%, entry 9). Next,

51 the impact of altering the amounts of fac-Ir(ppy)3 and Hantzsch ester (HE) were investigated. The role of HE is to reduce the oxidized fac-Ir(ppy)3) back to ground state and a combination of 2 mol% of photocatalyst and 0.5 eq of HE was found to increase the yield to 46% (entry 12). However, in a control experiment, omitting the photocatalyst in order to investigate its role in the reaction, 14a was formed in 49% yield (entry 15). This result was highly encouraging, due to the prohibiting cost of fac-Ir(ppy)3 and the realization that efficient generation of the alkyl radical was possible using solely Pd(PPh3)4 and the low-energy irradiation. Further optimization targeted the solvent volume and amount of reactants used. Here, diluting the reaction mixture improved the yield (64%, entry 16) while increasing the amount of 12 to 0.3 mmol led to an optimal concentration (71%, entry 17). Lastly, the organic solvent was altered to either toluene (entry 18) or anisole (entry 19), solvents that have previously been used in carbonylative Suzuki-Miyaura couplings.31,262– 264 Use of an aromatic solvent seemed necessary as experiments with acetonitrile and 1,4-dioxane had proven unsuccessful (entries 4 and 14). Benzene and related aromatic hydrocarbons have been shown to act as photosensitizers in the decomposition of alkyl iodides, which might be the function of the aromatic solvent in this reaction as well.265 As toluene also has been used as a substrate for C-H functionalization under photoredox conditions,266 the conditions as in entry 17 were selected for further evaluation.

Investigation of the Reaction Scope The scope of the reaction with respect to the alkyl halide component was explored and the results are presented in Table 6. Alkyl iodides generally performed well, with the yields ranging from 50% to 71%. No reactivity trend could be discerned between primary and secondary alkyl iodides but tertiary iodides were significantly more challenging. Compound 14n was only formed in trace amounts even after a prolonged reaction time whereas the adamantane derivative 14k formed in 61% yield. Of note is the isolation of tosyl-protected piperidine derivative 14e in 60% yield while the Boc-protected analog did not form at all. The reaction showed excellent chemoselectivity for the iodo-substituted carbon over the chloro-substituted carbon in the synthesis of 14j (71%). In order to extend the substrate scope beyond unactivated alkyl iodides, even more challenging unactivated alkyl bromides were employed in the reaction (Table 6). Gratifyingly, products 15a, 15f–g, 15i–k and 15o–p were isolated from their respective brominated starting material albeit with lower yields ranging from 26% to 61%. The reactivity trend for the alkyl bromides was similar to that of the alkyl iodides, with primary and secondary alkyl bromides performing equally well and tert-butyl bromide not giving product at all. Another demonstration of the chemoselectivity of the reaction came in the synthesis of 15j, which was isolated in 61% yield. The double bond in 15p

52 was left untouched, in contrast to radical reactions with similar substrates.259,267 The lower yield of the products synthesized from the alkyl bromides can be explained in part by remaining alkyl bromide after 24 h of reaction. In- creasing the irradiation time consequently improved the yield, from 30% to 40% for 15f and from 41% to 48% for 15i. However, not all reactions displayed unreacted alkyl bromide and in another attempt to increase the yield, both reaction chambers were heated to 50 °C. This resulted only in a very modest rise, from 35% to 39% for 15a.

Table 6. Investigation of the reaction scope with alkyl halide substrates

a b c Reaction conditions from entry 17, Table 5. 1 mmol scale. 72 h reaction time. Crxn and CCO heated to 50 °C. d55 h reaction time.

53 The difference in reactivity for the primary and secondary alkyl halides compared to the tertiary alkyl halides might be attributed to the stability of the formed alkyl radicals. Tertiary radicals are more stable than their primary and secondary counterparts, but the rate of carbonylation is reversed, i.e. primary radicals have the highest carbonylation rate.268,269 tert-Butyl iodide was used as substrate in a carbonylative Suzuki-Miyaura coupling performed under high pressures of carbon monoxide (45 bar) and intense visible light/UV irradiation and 14n was formed in 48%.253 Since the starting alkyl halide is con- sumed, the reason for the unsuccessful reaction here might be attributed to the lower pressure of carbon monoxide (2.5 bar). To explore the boronic acid component, a set of different aryl boronic acids were used as substrates in the reaction (Table 7). A range of aryl boronic acids were found to be compatible with the reaction and were isolated in yields ranging from 26% to 83%.

Table 7. Investigation of the reaction scope with aryl boronic acid substrates

Reaction conditions from entry 17, Table 5. Synthesis of 18. I) KI, NaHCO3, toluene, 100 °C, a b 22 h, 55%. HCOOH/H2SO4 used instead of Mo(CO)6. 48 h reaction time.

54 Varying the electron density of the ring did not affect the product outcome, as seen by the yields of electron-deficient 16f–h and electron rich 16j. Although fluoro- and chloro-substituted 16f–h were isolated in good to very good yields, their bromine-analog, 16m was only formed in trace amounts even after a prolonged irradiation time. A slight sensitivity to steric hindrance was evidenced by ortho-substituted products 16a, 16e, 16i and 16l, which were isolated with somewhat lower yields than the para-substituted products. To demonstrate the applicability of the developed method, the atypical antipsychotic drug melp- erone (18) was synthesized with 16g as an intermediate. With the carbonylative Suzuki-Miyaura reaction developed herein, the synthetic route of melp- erone was shortened to two steps.

Proposed Catalytic Cycle A set of control experiments were performed (Table 8) in order to gain insight into the reaction mechanism and the role of the reaction components. Without a catalyst, 14a was not detected (entry 1) whereas trace amounts of product were detected in the absence of LED irradiation (entry 2). Addition of 1 equiv- alent of radical scavenger 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) to the reaction led to detection of the cyclohexyl-TEMPO adduct and no traces of product (entry 3). Exploring the conditions for cyclohexyl radical formation and omitting either Pd(PPh3)4 (entry 4) or LED irradiation (entry 5) while adding TEMPO led to no formation of 14a but the TEMPO-adduct was only detected when the reaction was performed without irradiation. In parallel to the reaction outcome in entries 2 and 3, 14a was not detected when cyclohexyl bromide reacted either without LED irradiation (entry 6) or with TEMPO (entry 7). Most, if not all, of the starting alkyl halide remained intact in the reactions performed. The control experiments point to the importance of both Pd(PPh3)4 and LED irradiation for quantitative product formation as only trace amounts of 14a did form when the reaction was performed in the dark (entry 2, Table 8 vs entry 17, Table 5). The need for both a palladium-catalyst and visible-light irradiation is further supported by the notion that most of the starting material remains unreacted when either component is missing. Their roles might be related to efficient generation of the alkyl radical as the presence of a cyclohexyl radical was detected when TEMPO was added to the reaction mixture. However, the remainder of unreacted cyclohexyl halide when TEMPO has been added is intriguing and implies an involvement of the alkyl radical itself in the propagation of the reaction.

55 Table 8. Control experiments for the carbonylative Suzuki-Miyaura reaction

Entry X Catalyst Additive Irradiation Starting mate- Product Adduct for- (1 equiv) rial left formeda mationb 1 I - - LED Yes No -

2 I Pd(PPh3)4 - - Yes Trace -

3 I Pd(PPh3)4 TEMPO LED Yes No Yes 4 I - TEMPO LED Yes No No

5 I Pd(PPh3)4 TEMPO - Yes No Yes

6 Br Pd(PPh3)4 - - Yes No -

7 Br Pd(PPh3)4 TEMPO LED Yes No Yes Reaction conditions: As in entry 17, Table 5. aOutcome determined by GC/MS- and/or LC/MS-analysis. bDetermined by LC/MS-analysis.

Based on the control experiments and the literature, a catalytic cycle through which the reaction can operate is presented in Figure 17. An initial, key step is suggested to be the generation of the alkyl radical, A, through an interplay between the palladium-catalyst and the visible-light irradiation. The role of Pd(PPh3)4 may be either as a photosensitizer, to promote formation of the radical,270 or via an electron transfer.10–14 In both cases, a palladium(I)-complex, B, is proposed to form in connection to A. The reaction can then proceed by two different pathways, which have been proposed to operate in carbonylations performed at high pressures of carbon monoxide250–253,271 (path I) or at ambient pressures of carbon monoxide248,256,272 (path II). In path I, A will react with carbon monoxide to acyl radical C which in turn reacts with B to form acylpalladium(II) complex D. Intermediate C can also propagate the radical formation by halide atom abstraction to generate acyl halide E which can enter the catalytic cycle. In path II, A and B instead forms a caged radical pair which has been proposed to either collapse to an alkylpalladium(II) complex F where the alkyl group can migrate onto a palladium-bound carbon monoxide or A can directly add onto a palladium-bound carbon monoxide molecule. As the cyclohexyl radical was detected in the control experiments, A must have escaped and re- joined the radical cage for this pathway to be plausible.273 Both pathways con- verge on D, from which a transmetallation with aryl boronic acid gives palladium(II) complex G. A reductive elimination furnishes product H and regenerates palladium(0). The experiments with radical scavenger TEMPO support the reaction proceeding through path I. The notion that most of the starting halide remained unreacted in the control experiments and that the removal of carbon monoxide

56 resulted in unreacted 12 also supports path I. These findings suggests that C is important for radical propagation. However, the absence of carbon monoxide might lead to collapse of A with the halide atom and reformation of starting material. Thus, more experiments are needed to fully elucidate whether path I or II is under operation in this reaction.

Figure 17. Plausible catalytic cycle. Adapted from paper III.

Synthesis of 11C-Labelled Ureas by Palladium(II)- Mediated Oxidative Carbonylation (Paper IV) Ureas can form as side products in transition metal-mediated aminocarbonylations, especially if a palladium(II)-precatalyst is used.103 However, the urea functional group is found in many biologically active compounds and is used in medicinal chemistry for its ability to modulate physicochemical properties and to offer unique binding modes, including the possibility of hydrogen bonding.274 Traditionally, [11C]urea and 11C-labeled urea derivatives have been synthesized from either [11C]phosgene or [11C]cyanide but these methods are associ- ated with long synthesis times, because of many chemical transformations, 275–277 11 and low Am. Methods utilizing [ C]carbon dioxide have been developed and are characterized by short synthesis times but these methods still suffer 278,279 from low Am because of the natural occurrence of carbon dioxide in air. 11 Here, [ C]carbon monoxide offers a theoretical improvement in Am because

57 of the low concentration of atmospheric carbon monoxide. Consequently, methods utilizing [11C]carbon monoxide together with selenium,89 rhodium(I)88,108,280 or palladium(II)281 have been presented. Selenium and palladium(II) can use amines as sole precursors whereas rhodium(I) necessitates the use of an azide in addition to the amine. The single report using selenium found the scope to be limited compared to reactions utilizing [11C]phosgene whereas the palladium(II)-mediated synthesis did not isolate any 11C-labeled ureas, thus making it difficult to evaluate the utility of the palladium-mediated method.

Aim The main objective of the study was to develop a robust and facile method to access various 11C-labeled ureas, based on a palladium(II)-mediated oxidative carbonylation (Figure 18). The use of [11C]carbon monoxide would allow some of the issues with related methods to be addressed, such as complicated production of labeling precursor and low Am.

[11 C]CO

O RNH2 [Pd] R 11 C R' N N R' R'' H N R'' H

Figure 18. Schematic reaction set-up for the synthesis of 11C-labeled ureas.

Synthesis of 11C-Labeled N,N’-Disubstituted Ureas Symmetrically substituted ureas are not found to the same extent in medicinal chemistry as their unsymmetrically substituted counterparts. However, examples of bioactive, symmetric ureas can be found in literature, such as ureas of [(7-amino(2-naphthyl)sulfonyl]phenyl amines which are active at the insulin receptor tyrosine kinase or sulfonylated naphthyl urea derivatives, inhibitors of protein arginine methyltransferases.282,283 Exploration of incorporation of carbon-11 into symmetrically substituted ureas turned out to be rather straightforward as the reaction with benzyl amine 19 and dichloro[9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene]-palla- 11 11 dium(II) (Pd(Xantphos)Cl2) only formed [ C]N,N’-dibenzylurea C-20a.

58 Modifying the reaction time and temperature to increase the [11C]CO-conversion led to the conditions described in Table 9, where a small scope for the synthesis of N,N’-disubstituted 11C-labeled ureas are presented. The RCY was based on the starting activity of [11C]carbon monoxide transferred to the reaction vial and the activity of the 11C-labeled product isolated after semi-preparative HPLC (decay-corrected). The reaction performed well with primary, aliphatic amines, returning RCYs of isolated products 11C-20a–c in the range 40%–65% and RCPs > 99%. Of note is the isolation of 11C-20c in 48% RCY as N,N’-dicyclohexylurea is an inhibitor of enzyme soluble epoxide hydrolase (sEH), another example of a bioactive symmetric urea.274 To investigate whether the 10 min reaction time led to a loss in product activity due to decay, 11C-20a was isolated after a 5 min reaction as well. The RCY for the 5 min reaction was lower compared to the 10 min reaction (41% and 65% respectively). However, as the RCYs are decay-corrected, the activities of 11C-20a at the end of synthesis were also compared. Thus, following a 5 min reaction, 11C-20a was isolated with 0.44 GBq after 34 min from the end of cyclotron irradiation (end of bombardment, EOB). From the 10 min reaction, 11C-20a was isolated with 0.83–0.88 GBq after 33–36 min from EOB.

Table 9. Scope of N,N’-disubstituted 11C-labeled ureas

Reaction conditions: Amine (30 µmol), Pd(Xantphos)Cl2 (4 µmol), THF (400 µL). The presented RCYs are the mean of two experiments. > 99% RCP. a5 min reaction time, single experiment.

In contrast to the aliphatic amines, aniline was a poor substrate and 11C-20d was only isolated in 4% RCY. This sluggishness has been suggested in a palladium(II)-mediated reaction and in methods utilizing [11C]carbon dioxide although no 11C-labeled aromatic ureas were isolated.281,284,285 However, the use of rhodium(I) allowed a number of 11C-labeled aromatic ureas to be isolated in good yields, albeit with an aromatic azide as the other aryl source.280

59 Synthesis of 11C-Labeled N,N’,N’-Trisubstituted Ureas In contrast to the symmetrical ureas, unsymmetrical ureas are more plentiful in biologically active compounds.274,286 Examples of unsymmetrically substituted ureas are found in inhibitors of sEH, derived from N,N’-dicyclohexyl urea, and in protein kinase inhibitors, aimed at various kinases. Optimization of the reaction aimed at the synthesis of 11C-labeled N,N’,N’- trisubstituted ureas is shown in Table 10, starting from the same conditions as in the optimization of the symmetric 11C-labeled ureas but with the addition of a secondary amine (piperidine, 21). The initial conditions led to a [11C]CO- conversion of 53% and a selectivity of 79%, thus estimating the RCY to be 42% for unsymmetrical 11C-22a (entry 1). The ratio of formation between 11C- 20a and 11C-22a was 12:88, a ratio that remained rather constant throughout the optimization experiments.

Table 10. Optimization of reaction conditions for synthesis of N,N’N’-trisubstituted 11C-labeled ureas

Entry Pd-source T (°C) [11C]CO-con- 11C-22a RCYc (%) versiona (%) selectivityb (%) 1 Pd(Xantphos)Cl2 120 53 ± 6 79 ± 3 42 ± 6 (3) 2 Pd(PPh3)2Cl2 120 69 ± 4 46 ± 4 32 ± 3 (3) 3 Pd(OAc)2 + dppf 120 95 ± 4 13 ± 4 12 ± 4 (2) 4 Pd(OAc)2 + dppp 120 75 ± 3 21 ± 1 16 ± 1 (2) 5 Pd(OAc)2 + 120 67 ± 9 10 ± 1 7 ± 2 (2) Xantphos 6 Pd(PPh3)4 120 93 - - (1) d 7 Pd(Xantphos)Cl2 120 43 ± 2 49 ± 4 21 ± 2 (3) 8 Pd(Xantphos)Cl2 80 57 ± 9 44 ± 7 26 ± 9 (3) 9 Pd(Xantphos)Cl2 150 44 ± 11 87 ± 4 41 ± 6 (4) e 10 Pd(Xantphos)Cl2 120 46 ± 4 63 ± 2 29 ± 3 (3) f 11 Pd(Xantphos)Cl2 120 58 ± 2 71 ± 4 42 ± 3 (3) g 12 Pd(Xantphos)Cl2 120 67 ± 2 89 ± 3 60 ± 3 (3) Reaction conditions: 19 (30 µmol), 21 (30 µmol), catalyst ([Pd] 4 µmol + 4 µmol ligand), THF (400 µL). 5 min reaction time unless otherwise stated. aPercentage of [11C]CO converted into non-volatile products. Decay-corrected. bPercentage of product formed, based on analytical HPLC of crude reaction mixture. cRadiochemical yield, estimated from the [11C]CO-conversion and product selectivity. Number of experiments in brackets. dDMF (400 µL) as solvent. eTwo equiv of 21. fFive equiv of 21. g10 min reaction time.

First, the palladium(II)-source was changed in order to improve the RCY (entries 2-5). A monodentate ligand along with cis-coordinating bidentate ligands were tested in addition to adding the trans-coordinating Xantphos sepa-

60 rately.287 Notably, while the [11C]CO-conversion was improved for all palladium salts tested (including the in situ formed palladium-complex with Xantphos), the product selectivities was markedly lower and did not result in improved RCYs. Performing the reaction with a palladium(0)-source was del- eterious for product formation although the [11C]CO-conversion was excellent (93%, entry 6). A change in solvent, from tetrahydrofuran to N,N-dimethylformamide, led to a drop in both [11C]CO-conversion and product selectivity (entry 7). Altering the reaction temperature had opposite effect on the parameters investigated. Lowering to 80 °C raised the [11C]CO-conversion slightly, compared to the conditions in entry 1, whilst product selectivity was markedly lost (entry 8). Raising the temperature to 150 °C, on the other hand, resulted in a loss of [11C]CO-conversion while improving the product selectivity (entry 9). The resultant RCY of 41% was the same as that obtained with 120 °C in entry 1 and as no improvement was made, the temperature was kept at 120 °C. Next, the stoichiometry between 19 and 21 was investigated. Increasing the amount of 21 however, did not improve the reaction further. Using two equivalents of 21, had a negative impact on both the [11C]CO-conversion and the product selectivity (46% and 63% respectively, entry 10) whereas using five equivalents of 21 returned a slightly improved [11C]CO-conversion (58%, entry 11). The ratio of formation between 11C-20a and 11C-22a was also improved, with almost quantitative formation of 11C-22a (2:98). However, the RCY was still only estimated to 42% because of a loss in product selectivity (71%). As there were no added advantages with using five equivalents of 21, the 1:1 ratio of the starting amines was kept. In a final experiment, the reaction time was increased to 10 min, which had been successful for the synthesis of 11C-20a. Here, both the [11C]CO-conversion and the product selectivity were improved, thus resulting in a RCY of 60% (entry 12). The scope for synthesis of N,N’,N’-trisubstituted 11C-labeled ureas was explored with the conditions as in entry 12, Table 10. Again, primary aliphatic amines performed well and ureas 11C-22a–d were isolated in RCYs ranging from 12% to 41% (Table 11). The improvement in RCY and isolated activity when performing the reaction for 10 min compared to 5 min was demonstrated for 11C-22a. When the reaction was run for 5 min, 0.15 GBq of 11C-22a was isolated 37 min after EOB, which corresponds to a RCY of 17%. For the 10 min reaction, the RCY was 41% and 11C-22a was obtained in 0.47–0.57 GBq 36–45 min after EOB. Compared to benzylamine and butylamine, 2-(2-ami- noethyl)pyridine proved to be a tough substrate and 11C-22c was isolated in 12% RCY. Urea 11C-22d, where steric hindrance had been introduced near the nitrogen, was isolated in a similar RCY (14%). The RCYs of aniline derivatives 11C-22e–h were, similar to the synthesis of 11C-20d, lower than the RCYs of aliphatic derivatives 11C-22a–d and the RCYs ranged from 5% to 9%. Urea 11C-22e was isolated in 7% RCY and 80% RCP, due to a trouble- some impurity. Both parameters were improved, to 12% and 97% respectively, with the use of three equivalents of aniline.

61 Table 11. Scope for synthesis of N,N’N’-trisubstituted 11C-labeled ureas

Reaction conditions as in entry 12, Table 10. The presented RCYs are the mean of two experiments unless otherwise stated. 21 was not added in the synthesis of 11C-22i, 11C-22j (aniline was used as the second amine) and 11C-22l. > 97% RCP unless otherwise stated. a5 min reaction time, single experiment. bAverage of three experiments, 80% RCP. cThree equiv of aniline. dAverage of three experiments. e90% RCP.

The functional group tolerance was explored with three para-substituted aniline derivatives (11C-22f–h). The fluoro-substituted 11C-22f and methoxy-substituted 11C-22h performed equally well with RCYs of 8%–9% whereas the nitro-substituted 11C-22g was isolated with a slightly lower RCY of 5%. Alt- hough the [11C]CO-conversion in general was > 60% and the product selectivities ranged from 33% to 71% for 11C-22e–h, the RCYs were below 10%. This points to a limitation in estimating the RCY from only the [11C]CO-conversion and the HPLC analysis of the crude reaction mixture (product selectivity). Cyclic urea 11C-22i was only isolated in 1% RCY and a RCP of 90%. 11C- Labeled sulfonylureas have been synthesized from the corresponding sulfonyl azide and an amine, in a reaction mediated by rhodium(I).88 Here however, sulfonylureas 11C-22j and 11C-22k were only formed in trace amounts starting from p-tolyl sulfonamide and an amine. Their formation was found to be independent on the amine used, as neither aniline (11C-22j) nor piperidine (11C- 22k) furnished isolable product.

62 2-Ethylisoindolin-1-one (22l) and similar lactams have been readily synthesized under palladium(II)-catalysis and atmospheric pressures of carbon monoxide.41 Under the present conditions, namely the low amount of [11C]carbon monoxide present and the short reaction time, N-ethylbenzyl amine proved to be a challenging substrate and 11C-22l was not formed at all. Instead, [11C]N-benzyl-N-ethylbenzamide formed as a result of scrambling of a phenyl group from the ligand to palladium. The identity of the side product was confirmed by coupling of benzoic acid and N-ethylbenzyl amine and comparing HPLC retention times. Addition of different alcoholic additives to the reaction mixture has been found to increase the conversion of ammonium cyanate to urea and the conversion of ammonium [11C]isocyanate to [11C]urea.276,288 To investigate if the addition of an alcoholic additive could improve the RCY in the present reaction, a set of selected 11C-labeled ureas were re-synthesized with the addition of 1-butanol (Table 12). The RCYs of aniline derivatives 11C-22f and 11C-22h were indeed improved (left, Table 12), from 8% and 9% to 28% and 21%, respectively. However, the syntheses of 11C-22e and 11C-22g were not affected (right, Table 12). Likewise, the RCYs of 11C-22a, 11C-22b and 11C-22i were not improved by the addition of 1-butanol.

Table 12. Exploring the effect of 1-butanol addition

Reaction conditions as in entry 12, Table 10, with the addition of 10 equiv of 1-butanol. Reac- tions performed as single experiments. RCP > 98% unless otherwise stated. aRCP 88%.

Judging by the successful outcome for 11C-22f and 11C-22h as well as the lack of improvement for 11C-22a and 11C-22b, the addition of 1-butanol seemed most beneficial when a poorly nucleophilic amine was used. However, the results for 11C-22e and 11C-22g, did not support a theory of nucleophilicity and further experiments are needed to elucidate the reason for the (selective) improvement. To demonstrate the feasibility of the method, in terms of applicability and 11 Am, sEH inhibitor C-26 was synthesized (Scheme 5). Synthesis of precursor 25 started from 4-hydroxypiperidine, which was Boc-protected to afford 23.

63 A Mitsunobu reaction furnished intermediate 24, which gave the secondary amine 25 after an acid-mediated deprotection.

Scheme 5. Synthesis of precursor 25 and 11C-26. I) di-tert-Butyl carbonate, 10% NaOH (aq.), THF, rt, 19 h, 95% II) Phenol, PPh3, DIAD, THF, rt, 18 h, 59% III) TFA, CH2Cl2, rt, 2.5 h, 96% IV) 2,4-Dichlorobenzyl amine, Pd(Xantphos)Cl2, THF, 120 °C, 10 min, 41 ± 7% RCY (average of three experiments, decay-corrected).

The oxidative 11C-carbonylation utilized 25 and 2,4-dichlorobenzyl amine fur- 11 nishing C-26 in 41% RCY and excellent RCP (> 99%). The Am was determined in two experiments and when starting from 17.8 GBq of [11C]carbon 11 monoxide, 1.9 GBq of C-26 was isolated 43 min from EOB, giving an Am of 247 GBq/µmol. The second experiment furnished an Am of 319 GBq/µmol when starting from 12.8 GBq of [11C]carbon monoxide and giving 2.1 GBq of 11C-26 at 41 min from EOB.

Proposed Reaction Mechanism Urea formation has been proposed by Hiwatari et al. to proceed through a carbamoyl-palladium species.289 Thus, hypothesizing that the 11C-labeled carbamoyl palladium species A was present during the reaction here, a number of paths through which the reaction can proceed are presented in Figure 19. In paths I and II, an 11C-labeled isocyanate C is formed by a base-assisted deprotonation (path I) or by a beta elimination (path II). Palladium(0)-complex B directly forms in path I, whereas reductive elimination of HCl furnishes the complex in path II. The formation of an isocyanate precludes the use of secondary amines as sole precursors in the reaction. However, in paths III–V, a secondary amine would be a feasible reaction substrate. Carbamoyl chloride E is proposed to form after a reductive elimination in path III, where attack of a second amine furnishes product. In paths IV and V, the second amine is proposed to react with A, where direct attack at the carbonyl carbon gives product in path IV and an initial coordination followed by reductive elimination takes place in path V.

64 B RNH 2 Pd0 P P O R 11 C N RNH2 HCl C HCl RNH2 I H Cl PdII C II P P D [11 C]CO O O HCl O Cl R E II 11 III 11 11 Pd N C R C R C R P P RNH2 PdII N Cl N N Cl H P P H Cl B RNH2 H H A O IV R 11 C N PdII H P Cl P RNH HCl B V 2 O F R 11 RNH2 C N PdII H P P RHN HCl B Figure 19. Proposed reaction paths in 11C-labeled urea formation. Adapted from paper IV.

Hence, the formation of the carbamoyl-palladium complex A allows the utilization of both primary and secondary amines, but experimental evidence supports an isocyanate intermediate, as in paths I and II.289–291 Furthermore, the formation of the isocyanate intermediate as proposed in path I is supported by kinetic studies.289 The observations made during the work on paper IV also largely support the reaction proceeding through an 11C-labeled isocyanate. Accordingly, performing the reaction with piperidine did not furnish the tetra-substituted 11C- labeled urea. The low RCY of 11C-20d (4%, Table 9) can be rationalized by path I, where the weakly basic aniline must deprotonate A to form [11C]phenyl isocyanate. Furthermore, [11C]4-fluorophenyl isocyanate was detected when the reaction was performed with only 4-fluoroaniline and Pd(Xantphos)Cl2. Subsequent addition of 21 gave product 11C-22f. However, the mere presence of an 11C-labeled isocyanate does not preclude the reaction from proceeding through another path. Thus, the low RCYs of aniline derivatives 11C-22e–h cannot be explained by the poor basicity of the aniline derivatives, as 21 should be able to both depronate A and attack C to yield product. The lower nucleophilicity of the aniline derivatives should hamper not only attack on C but also the association to the palladium complex, thus the formation of A. DFT (density functional theory) calculations were thus performed with carbon monoxide, methyl amine and Pd(Xantphos)Cl2 to study the energetic feasibility of path I. Indeed, was path I found to be viable and in Figure 20, selected intermediates and their free energies are shown. Carbamoyl-palladium complex Ib represents the most stable intermediate from which there is an

65 energy barrier of 33.4 kcal/mol to form the isocyanate through the base-assisted deprotonation.

Figure 20. Calculated free energies of selected intermediates in path I. Adapted from paper IV.

Acylamidines by Pd-Catalyzed Aminocarbonylation: One-Pot Cyclizations and 11C-Labeling (Paper V) Acylamidines are known intermediates in the synthesis of various heterocycles, such as 1,2,4-triazoles,292 1,3,5-triazines293 and 1,2,4-oxadiazoles294 but the acylamidine motif is also found in biologically active compounds, such as angiotensin II receptor ligands,295 renin inhibitors296 and thrombin inhibitors.297 Various strategies have been adopted for the synthesis of acylamidines, with an acylation of amidines already reported in 1889.298 Other approaches have included reactions of acylimidates and amines,299 hydroalumination of cyanamides300 and transition metal-catalyzed transformations of N-sulfonyl- 1,2,3-triazoles301 and O-propargylic oximes.302 These reactions feature spe- cialized substrates but a more general method was reported in 2011, when a palladium(0)-catalyzed carbonylation of aryl iodides and amidines furnished acylamidines as intermediates en route to 1,2,4-triazoles.292 As the study was focused on synthesis of 1,2,4-triazoles, only one acylamidine product was isolated and characterized.

66 Aim The main objective of the study was to develop a method for the synthesis and isolation of acylamidines. Performing the reaction in a double-chamber system would allow the use of a solid source of carbon monoxide. To extend the applicability of the method, one-pot cyclizations to different heterocycles and 11C-labeling was also pursued (Figure 21).

Figure 21. Schematic reaction set-up for the synthesis of 11C/12C-acylamidines and their subsequent cyclization.

Acylamidine Synthesis and One-Pot Cyclizations An optimization was performed in order to find reaction conditions suitable for the palladium(0)-catalyzed formation of acylamidines. Screening of solvents, catalyst loading and addition of ligand resulted in the conditions described in Table 13, where the results of selected acylamidines are presented. The original conditions featured triphenylphosphine (PPh3) as the ligand, which was very productive in the synthesis of 27a and 27b but less productive in the synthesis of 27c–f. Synthesized from electron-poor aryl iodides, the yields of 27c and 27d were improved with the use of Xantphos as ligand. Xantphos also returned a higher yield for 27e, synthesized from an electron- rich aryl iodide, whereas DPEphos gave the highest yield for sterically encumbered 27f. The amidines were evaluated with DPEphos, which returned electron-neu- tral and electron-rich 28a–c in good yields (71%) whereas 28d and 28e, synthesized from electron-poor amidines, were isolated in lower yields (45% and 41%, respectively). Here, both triphenylphosphine and 1,1’-bis(diphenylphosphino)ferrocene (dppf) improved the yields.

67 Table 13. Selected acylamidines

Reaction conditions: Chamberrxn: Aryl iodide (0.5 mmol), amidine (1.5 equiv), Pd(OAc)2 (5 mol%), NEt3 (2.5 equiv), DMF (2.5 mL). ChamberCO: Mo(CO)6 (0.5 equiv), DBU (2.5 equiv), a b c d 1,4-dioxane (2.5 mL). PPh3 (10 mol%). Xantphos (5 mol%). DPEphos (5 mol%). dppf (5 mol%).

One-pot cyclizations to form 1,2,4-oxadiazoles and 1,2,4-triazoles were next pursued. The 1,2,4-oxadiazoles were prepared by addition of hydroxylamine hydrochloride to give 29a–d whereas addition of a hydrazine-derivative furnished 1,2,4-triazole 30a (Scheme 6).292,294,303

Scheme 6. Synthesis of 1,2,4-oxadiazoles 29a–d and 1,2,4,-triazole 30a. I) See reaction conditions in Table 13. Ligand: 29a–b, 30a used DPEphos (5 mol%). 29c–d used Xantphos (5 mol%) II) NH2OH·HCl, AcOH (aq.), 120 °C, MW, 20 min III) 4-methoxyphenylhydrazine, AcOH, 80 °C, 1h.

11C-Labeled Acylamidines and One-Pot Cyclization An advantage of the carbonylation reaction is the ability to incorporate carbon isotopes, thus the incorporation of [11C]carbon monoxide into acylamidines was pursued to expand the scope of 11C-labeled functional groups and to be an aid in the development of future PET tracers. A small set of conditions suitable for incorporation of [11C]carbon monoxide were investigated by using 4-iodoanisole 31a and benzamidine 32 as substrates (Table 14).

Table 14. Screening conditions for incorporation of [11C]carbon monoxide

[11C]CO- Product RCYc Entry Pd-source Ligand Solvent conversiona selectivityb (%) (%) (%) 1 Pd(OAc)2 Xantphos DMF 98 ± 0 49 ± 9 48 ± 9 2 Pd(OAc)2 PPh3 1,4-dioxane 91 ± 4 56 ± 7 49 ± 6 3 Pd(PPh3)4 - 1,4-dioxane 80 ± 5 67 ± 4 54 ± 6 4 Pd(PPh3)4 - DMF 54 ± 4 87 ± 5 47 ± 6 Reaction conditions: 31 (9 µmol), 32 (18 µmol), Pd(OAc)2 (4.5 µmol), PPh3 (9 µmol), Pd(PPh3)4 (0.9 µmol), NEt3 (36 µmol), solvent (400 µL). Experiments performed in triplicate. aPercentage of [11C]CO converted into non-volatile products. Decay-corrected. bPercentage of product formed, based on analytical HPLC of crude reaction mixture. cRadiochemical yield, estimated from the [11C]CO-conversion and product selectivity.

69 Utilizing Pd(OAc)2 and Xantphos while running the reaction at 120 °C for 10 min in N,N-dimethylformamide gave 98% [11C]CO-conversion and 49% product selectivity, thus resulting in an estimated RCY of 48% for 11C-27e (entry 1). The aim was then to raise the product selectivity while maintaining the [11C]CO-conversion. Changes in palladium source, ligand and/or solvent managed to improve the product selectivity (56%-87%, entries 2-3). The best RCY was obtained with Pd(PPh3)4 (54%, entry 3). The applicability of the method was next explored by the synthesis and isolation of three representative 11C-labeled acylamidines (Table 15). The RCYs were based on the amount of [11C]carbon monoxide transferred to the reaction vial and the activity of the isolated 11C-labeled product. Electron-rich 11C-27e was isolated in 24% RCY. To evaluate the effect of reaction time on the RCY and activity of the isolated product, 11C-27e was also isolated after a 5 min reaction which gave 83 MBq of 11C-27e 37 min from EOB (RCY 7%, Table 15). The 10 min reaction gave 190 MBq of 11C-27e 41 min from EOB. Electron-poor 11C-27g was thereafter isolated in a gratifying 36% RCY whilst sterically encumbered 11C-27f did not form at all. Modifying the reaction conditions by change of solvent, temperature or ligand did not lead to formation of 11C-27f either.

Table 15. 11C-Labeled acylamidines

Reaction conditions as in entry 3, Table 14. The presented RCYs are the mean of three experiments for 11C-27e and four experiments for 11C-27g. RCP > 95%. a5 min reaction time, single experiment.

The encouraging results of the 4-substituted aryl iodides were followed by determination of the molar activity for 11C-27g. When starting from 14.3 GBq of [11C]carbon monoxide, 1.7 GBq of 11C-27g was isolated 38 min from EOB thus returning an Am of 488 GBq/µmol. In the second experiment the Am was 650 GBq/µmol, when starting from 15.4 GBq of [11C]carbon monoxide and isolating 2.1 GBq of 11C-27g 38 min from EOB. These encouraging results 11 are amongst the highest Am obtained for the C-labeled compounds presented in this thesis.

70 The one-pot carbonylation/cyclization developed for the 12C-reaction was also pursued for the synthesis of a 11C-labeled 1,2,4-oxadiazole. In Scheme 7, the synthesis of 11C-29a from 4-iodotoluene 31b and 32 is presented. Sodium hypochlorite and hydroxylamine hydrochloride were investigated for the cyclization but only hydroxylamine hydrochloride gave full consumption of the intermediate 11C-27a formed.294,303 Oxadiazole 11C-29a was isolated in 25% RCY and > 99% RCP.

11 11 Scheme 7. Synthesis of C-29a. I) Pd(PPh3)4, [ C]CO, NEt3, 1,4-dioxane, 120 °C, 5 min II) NH2OH·HCl, AcOH (aq.), 150 °C, 5 min, 25 ± 2% RCY (decay-corrected, average of two experiments).

Summary of Papers III–V Although the carbonylative toolbox is vast, especially for conventional palladium-catalyzed carbonylation reactions but also for 11C-carbonylations reactions, there are details to further explore. The palladium-mediated 11C/12C- carbonylations presented in papers III–V have aimed at synthesizing carbonyl derivatives found in biologically active compounds and have sought to refine and expand the settings and the scopes of the reactions. The work in paper III investigated the utilization of unactivated alkyl iodides in a carbonylative Suzuki-Miyaura coupling. A simplified reaction set- up and mild reaction conditions compared to analogous reactions were presented along with the extension to alkyl bromides as substrates. The use of a double-chamber system circumvented the use of carbon monoxide gas and the cheap and low energy-output LED lamps were proven to be enough to generate the key alkyl radicals together with Pd(PPh3)4. However, the combination of the results was perhaps the most exciting finding: that a simple palladium- catalyst, under visible-light irradiation, promoted efficient radical generation from unactivated alkyl halides to produce aryl alkyl ketones under ambient pressures of carbon monoxide. The 11C-carbonylation reactions in papers IV and V utilized simple and easily accessible starting materials and the practical utility of the methods was demonstrated by isolation of the 11C-labeled compounds and by the molar activity determinations. The palladium(II)-mediated synthesis of 11C-labeled ureas in paper IV utilized amines as sole precursors in addition to [11C]carbon monoxide, thus presenting a significant simplification in relation to methods utilizing other 11C-synthons in particular but also to other metal mediators. The method was found to be compatible with several functional groups and

71 DFT calculations supported the reaction proceeding through an 11C-labeled isocyanate. The palladium(0)-catalyzed reaction in paper V utilized a solid source of carbon monoxide and returned acylamidines in moderate to excellent yields. Different ligands were found to be productive for different substrates. The applicability of the method was further demonstrated by one-pot cyclizations to form heterocycles and 11C-labeling to give 11C-labeled acylamidines and a 11C-labeled 1,2,4-oxadiazole. Although the investigated scope was small, the results were promising and the synthesis of the 11C-labeled acylamidines and the 11C-labeled 1,2,4-oxadiazole is unprecedented in the literature, thus representing new ways to utilize [11C]carbon monoxide.

72 Concluding Remarks

The work presented in this thesis has utilized and explored palladium-mediated incorporation of carbon monoxide or [11C]carbon monoxide into different biologically relevant compounds, containing derivatives of the carbonyl functional group. The reactions are summarized in Figure 22, which also exempli- fies the versatility possible to attain via the carbonylation reaction. The work presented here has added knowledge regarding the structure-activity relationship for ligands aimed at VAChT and corroborated the expediency of a general method for 11C-labeling in tracer development. Furthermore, the work has contributed to methods for the synthesis of isotopically unmodified and/or 11C-labeled carbonyl derivatives, including unprecedented 11C-labeled products.

Figure 22. The 11C- and 12C-carbonylation reactions presented in the thesis.

The key findings and conclusions from papers I–V are summarized below:

 The work in papers I and II described the early stages of PET tracer development in the quest of finding a PET tracer for VAChT. The benzovesamicol scaffold was functionalized with an amide, and

73 further modified to probe the binding to VAChT. The most promising results were obtained with substitution on the C5-position on the tetrahydronaphthol ring, although no ligand was found suitable for further preclinical evaluation. However, the N-benzyl derivatives displayed interesting features in terms of affinity and selectivity and thus represents viable alternatives for future VAChT PET tracer development.

 The use of visible-light irradiation together with palladium catalysis enabled the carbonylation of unactivated alkyl iodides and alkyl bromides described in paper III. The reaction was performed under ambient temperature and pressure whilst utilizing a solid source of carbon monoxide and represents a simplified and accessible reaction set-up.

 The radiochemical toolbox was further expanded by the work described in papers IV and V. Utilization of a palladium(II)-source, together with amines and [11C]carbon monoxide resulted in a simple and robust method for the synthesis and isolation of 11C-labeled ureas in paper IV. A palladium(0)-catalyzed synthesis of acylamidines was presented in paper V together with a method for their 11C-labeling. The applicability of the methods was further demonstrated by the one-pot cyclizations to give both isotopically unmodified and 11C-labeled heterocycles.

74 Acknowledgments

My PhD studies were funded by the disciplinary domain of Medicine and Pharmacy at Uppsala University. The work performed during these years has come through with contributions from different persons. Persons who in different ways have helped me in my work and with the realization of this thesis.

First, I would like to thank my supervisors. Mats Larhed, for your confidence in me and for always being available with answers and advices to all sorts of queries, both chemistry and non-chemistry-related. Gunnar Antoni, for introducing me to the world of radiochemistry and for always keeping a positive outlook. Jonas Eriksson, for introducing me to the xenon system and the in- tricacies of 11C-carbonylations. I have learnt a great deal by our joint work and by our discussions. Luke Odell, for your encouragement. Your scientific curi- osity is inspiring and our discussions and collaborations have taught me a lot.

My co-authors and collaborators; Sergio Estrada, Håkan Hall, Rashidur Rah- man and Marie Svedberg for the preclinical work with the ligands in paper I. Alf Thibblin, for introducing me to 11C-carbonylations and the apparatus in hot cell 3. Andreas Blomgren and Fredrik Wångsell for your contributions to paper I. Maria De Rosa, for the help with the synthesis of the VAChT ligands in paper II. Peter Brust and Winnie Deuther-Conrad, for a great collaboration on the evaluation of the VAChT ligands from paper II. Peter Brandt, for taking your time to perform the DFT calculations in paper IV and for discussing them with me. Patrik Nordeman, for all the help in the hot cells, especially on the 11C-labeled ureas in paper IV. Alaa Abbas and Marc Stevens, for setting the foundations to paper V. Tamal Roy, for taking your time to perform the heterocyclic syntheses in paper V. Jonas Rydfjord, for all your hard work with the acylamidines in paper V.

The basis for the day-to-day work has been at OFK and I would like to thank past and present colleagues for making it an enjoyable place to work. The lunch-room spirit has been further lifted these last months by the company of colleagues from AFK and FKOG.

The personnel at the University Hospital PET centre, especially the chemistry division. I would like to thank all of you for taking your time to help me with

75 all sorts of things and for your company in the lunchroom and by the cyclotron control.

I have spent most of the time in the office B5:522d by myself but I have never been alone. Past and present colleagues on floor five, especially my nearest neighbours Johanna Larsson, Tamal Roy, Jonas Rydfjord, Marc Stevens, fel- low early bird Johan Wannberg and Ulrika Yngve, thank you for your company.

Sorin Srbu, Gunilla Eriksson and Birgitta Hellsing. You have always provided answers and solutions to all sorts of questions and problems, thereby greatly facilitating my work. Ulrika Rosenström, for valuable discussions regarding teaching and for always listening and being perceptive about teaching and teaching-related matters. Malin Graffner Nordberg, for your support during these years. I have appreciated our discussions and how you have made me look at (my) research from a different perspective.

Attendances to conferences in Sweden and abroad has been made possible by financial support from Apotekarsocieteten and OFK. These conferences would not have been as enjoyable without the company of; divisional colleagues whom I have visited Organikerdagarna in Gothenburg, Stockholm and Umeå with; Gunnar Antoni, Jonas Eriksson, Patrik Nordeman, Luke Odell, Anna Orlova, Stina Syvänen, Vladimir Tolmachev, Irina Velikyan and Ola Åberg for the ISRS conferences in Jeju, South Korea and Dresden, Germany; Jonas Rydfjord, Bobo Skillinghaug, and Fredrik Svensson for ULLA in Lon- don; (yelp-master) Rebecka Isaksson for ACS in San Francisco.

Jonas Rydfjord, Supaporn Sawadjoon and Marc Stevens, for taking your time to read this thesis and giving me valuable feedback.

My master student and SOFOSKO-student, Homan Hamdi and Doaa Hassan, thank you for your hard work in the lab.

Doing a PhD and writing a thesis are in many ways unique experiences and I have shared these experiences with many great PhD students. A special thanks to Anna Skogh and Linda Åkerbladh, whom I have shared most of the ups and downs with during these years. Hiba Alogheli, Anna Karin Belfrage, Karin Engen and Rebecka Isaksson for great company and many laughs. Jonas Rydfjord, for all sorts of discussions where those related to acylamidines and thesis writing have been especially helpful these last months.

Anne och Lars, för all hjälp ni ger oss i stort som smått.

76 Mamma och pappa, för att ni alltid stöttar mig, i allt vad jag tar mig för.

Märta, min älskade dotter. Allt annat spelar lite mindre roll jämfört med dig.

Robert, min älskade make. För att du alltid finns där för mig.

77 Appendix 1

Small Animal PET and Biodistribution Studies (Paper I) Small Animal PET Sprague-Dawley rats, weighing approximately 350 g, were used. Animal permission was granted by the local Research Animal Ethics Committee C 38/9. Before the PET study, the rats were anesthetized with isoflurane (approximately 3%) in a 40/60% mixture of oxygen and air. The labeled compound (11C-(±)4b 8.6 MBq injected, 11C-(±)4d 9.32 mBq injected and 11C-(±)4e 9.05 MBq injected) was administered as a bolus in the tail vein while being in the PET camera and PET data collection were simultaneously started and lasted 40 min. Imaging was performed on a GE Triumph animal PET/SPECT/CT scanner with a 10 cm transaxial and 7.5 cm axial field of view and a Hama- matsu SHR 7700 PET camera (Hamamatsu, Japan), with an aperture of 30 cm, field of view 12 cm. Breathing was monitored throughout the study. After the PET study, CT was run with the same Triumph animal PET/SPECT/CT scanner. The images were analyzed using the software PMOD (PMOD Technolo- gies Ltd., Zürich, Switzerland). Regions of interest were drawn on the fused PET/CT image or on the PET image alone, and time-activity curves (TACs) were generated for the whole brain.

Organ distribution studies In order to evaluate the biodistribution of 11C-(±)4b and 11C-(±)4d–e, the uptake of radioactivity in a number of rat organs was investigated. After the completion of the small animal PET studies, the rats were killed with an over- dose of pentobarbital in the tail vein. Some organs (blood, brain, liver, pan- creas, kidney, intestine (with contents)) were collected and weighed. The radioactivity in the organs was determined and corrected for radioactive decay in a well-type scintillation counter with background and dead-time correction. Organ values were calculated as standardized uptake value (SUV), as follows:

gBqACT )( SUV  gBWBqDOSE )()(

78 where ACT is the measured concentration of radioactivity corrected for physical decay (Bq/g), DOSE is the administered amount of radioactivity (Bq) and BW is the body weight of the animal (g).

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99 Acta Universitatis Upsaliensis Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 241 Editor: The Dean of the Faculty of Pharmacy

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