ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2016

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 219

Multicomponent Reactions in11C/12C Chemistry

– Targeting the Angiotensin II Subtype 2 Receptor

MARC STEVENS

ISSN 1651-6192ISBN 978-91-554-9636-4urn:nbn:se:uu:diva-295436

Dissertation presented at Uppsala University to be publicly examined in Hall B21, BMC,Husargatan 3, Uppsala, Friday, 23 September 2016 at 09:15 for the degree of Doctor ofPhilosophy (Faculty of Pharmacy). The examination will be conducted in English. Facultyexaminer: Associate Professor Neil Vasdev (Harvard Medical School).

AbstractStevens, M. 2016. Multicomponent Reactions in 11C/12C Chemistry. – Targeting theAngiotensin II Subtype 2 Receptor. Digital Comprehensive Summaries of UppsalaDissertations from the Faculty of Pharmacy 219. 93 pp. Uppsala: Acta UniversitatisUpsaliensis. ISBN 978-91-554-9636-4.

Section 1 of this thesis contains an introduction to method development in organic synthesis,multicomponent reactions, sulfonyl azides, tracer development in 11C chemistry and thebiological target.

Section 2 describes the use of sulfonyl azides in carbonylative chemistry. Paper I coversdevelopment of a diazotransfer protocol. In total, 30 arylsulfonyl azides were synthesised fromprimary sulfonamides (20–90% yield). 15N mechanistic studies were carried out and in PaperII, the products were converted into sulfonamides, sulfonylureas and sulfonyl carbamates (19–90% yield). For ureas and carbamates, a two-chamber protocol was employed to release COfrom Mo(CO)6. 15N mechanistic studies showed that the sulfonamides were formed by directdisplacement of azide.

Section 3 covers imaging and biological studies of the angiotensin II receptor subtype 2(AT2R). In Paper III, 12 11C-sulfonyl carbamates were prepared in isolated radiochemical yieldsof 3–51% via Rh(I)-mediated carbonylation. The first non-peptide AT2R agonist, C21, waslabelled (isolated RCY 24±10%, SA 34–51 GBq/µmol). C21 was tested in a prostate cancerassay, followed by biodistribution and small-animal PET studies. In Paper IV, a 11C-labelledAT2R ligand prepared via Pd(0)-mediated aminocarbonylation was used for autoradiography,biodistribution and small-animal PET studies.

Section 4 describes the development of a multicomponent method for the synthesis of 3,4-dihydroquinazolinones (Paper V). 31 3,4-dihydroquinazolinones were synthesized via a cycliciminium ion.

Keywords: carbonylation, positron emission tomography, multicomponent reactions, AT2R, 3,4-dihydroquinazolinone, sulfonyl azides

Marc Stevens, Department of Medicinal Chemistry, Box 574, Uppsala University, SE-75123Uppsala, Sweden.

© Marc Stevens 2016

ISSN 1651-6192ISBN 978-91-554-9636-4urn:nbn:se:uu:diva-295436 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-295436)

So long, and thanks for all the fish!

List of Papers

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

I Stevens, M. Y., Sawant, R.T., Odell, L. R.* (2014) Synthesis of

Sulfonyl Azides via Diazotransfer using an Imidazole-1-Sulfonyl Azide Salt: Scope and 15N NMR Labelling Experiments. Journal of Organic Chemistry, 79(11):4826–4831. Featured Article.

II Chow, S. Y., Stevens, M. Y., Odell, L. R.* (2016) Sulfonyl Az-ides as Precursors in Ligand-free Palladium-Catalyzed Synthesis of Sulfonyl Carbamates and Sulfonyl Ureas and Synthesis of Sul-fonamides. Journal of Organic Chemistry, 81(7): 2681–2691. Featured Article.

III Stevens, M. Y., Chow, S. Y., Estrada, S., Eriksson, J., Asplund, V., Orlova, A., Mitran, B., Antoni, G., Larhed, M., Åberg, O., Odell, L. R.* (2016) Synthesis of 11C-labelled Sulfonyl Carba-mates via a Multicomponent Reaction Employing Sulfonyl Az-ides, Alcohols and [11C]CO. Manuscript, submitted.

IV Åberg, O., Stevens, M. Y., Lindh, J., Wallinder, C., Hall, H., Monazzam, A., Larhed, M.,* Långström, B.* (2010) Synthesis and Evaluation of a 11C-labelled Angiotensin II AT2 Receptor Ligand. Journal of Labelled Compounds and Radiopharmaceu-ticals, 53: 616–624.

V Stevens, M. Y‡, Wieckowski, K‡., Wu, P., Sawant, R. T., Odell, L. R.* (2015) A Microwave-Assisted Multicomponent Synthesis of Substituted 3,4-Dihydroquinazolinones. Organic and Bio-molecular Chemistry, 13: 2044–2054. Also highlighted in 2015 Hot Articles in Organic and Biomolecular Chemistry.

Reprints were made with permission from the respective publishers. ‡Contributed equally to this work *Corresponding author

Author Contribution Statement

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

I Carried out method development, synthesised majority of final

compounds, prepared isotopically enriched compounds for mechanistic studies, collated experimental data and drafted the manuscript.

II Assisted in synthesis of precursors and final compounds, collated experimental data, prepared isotopically enriched compounds for mechanistic studies and assisted in manuscript preparation.

III Prepared all precursors and several analytical standards (majority prepared by second author), performed radiochemistry method development, prepared final labelled compounds and analytical standard, assisted in ex vivo biodistribution studies, collated ex-perimental data, and drafted the manuscript.

IV Prepared precursors and reference standards, assisted in manu-script preparation.

V Participated in method development, prepared majority of pre-cursors and final compounds together with third author, collated experimental data and drafted the manuscript.

Other Papers Not Included in This Thesis

VI Desroses, M., Wieckowski, K., Stevens, M. Y., Odell, L. R.* (2011) A Microwave-Assisted, Propylphosphonic Anhydride (T3P®) Mediated One-Pot Fischer Indole Synthesis. Tetrahedron Letters, 52(34):4417–4420.

VII Sawant, R. T., Stevens, M. Y., Odell, L. R.* (2015) 3,4-Dihydro-3-(2-hydroxyethyl)-4-(nitromethyl)quinazolin-2(1H)-one. Mol-bank, 2015: M866.

VIII Stevens, M. Y,* Rozycka-Sokolowska, E., Andersson, G., Odell, L. R., Marciniak, B., Joule, J. (2015) Cylic amidines as precur-sors for imidazoles. ARKIVOC, 2015, 5, 219–229.

IX Sawant, R. T., Stevens, M. Y., Odell, L. R.* (2015) Rapid Ac-cess to Polyfunctionalized 3,4-Dihydroquinazolinones through a Sequential N-Acyliminium Ion Mannich Reaction Cascade. Eu-ropean Journal of Organic Chemistry, 2015, 7743–7755.

X 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 via Visible-light Photoredox Catalysis. Chemistry- A European Journal, 2016, 22, 1–8. Featured on the front cover of the July 2016 issue.

XI Sawant, R. T, Stevens, M. Y., Odell, L. R.* (2016) Rapid Access to Diverse Molecular Scaffolds using a Microwave-Assisted Branching Cascade. Manuscript, submitted.

*Corresponding author

Contents

1 Introduction ................................................................................................ 13 1.1  Overview and Thesis Context ......................................................... 13 1.2 Introduction to Method Development in Organic Synthesis .............. 14 1.3 Multicomponent Reactions................................................................. 15 

1.3.1  The Mannich and aza-Henry Reactions- An Expedited Route to Polysubstituted 3,4-Dihydroquinazolinones ......................... 16 1.3.2  Palladium and Rhodium in Organic Synthesis ...................... 17 1.3.3  Carbonylation Reactions ........................................................ 19 

1.4  Sulfonyl Azides-Structure and Synthetic Applications ................... 22 1.5  Tracer Development in 11C Radiochemistry ................................... 25 

1.5.1  Positron Emission Tomography (PET) .................................. 25 1.5.2  Considerations in Tracer Development Using 11C ................. 27 1.5.3 Carbonylative Reactions in 11C Radiochemistry ........................ 30 

1.6  The Renin-Angiotensin System as a Biological Target .................. 32 1.6.1 The Angiotensin II Receptor Subtypes 1 and 2 (AT1R and AT2R)-Biology .................................................................................... 32 1.6.2 Nonpeptidic AT2R-Selective Compounds .................................. 33 

1.7  Access to 11C-labelled AT2R Agonists- Labelling Strategies ......... 35 

2  Sulfonyl Azides as a Versatile Entry Point to Novel Carbonylation Chemistry ...................................................................................................... 37 

2.1 Synthesis of Sulfonyl Azides via Diazotransfer using an Imidazole-1-Sulfonyl Azide Salt: Scope and 15N NMR Labelling Experiments (Paper I) .................................................................................................. 37 

2.1.1 Background and Aim .................................................................. 37 2.1.2 Adaptation of Existing Protocols ................................................ 38 2.1.3 Preparation of Structurally Diverse Sulfonyl Azides .................. 39 2.1.4 Mechanistic Studies .................................................................... 40 2.1.5 Summary and Future Outlook (Paper I) .................................... 42 

2.2 Sulfonyl Azides as Precursors in Ligand-free Palladium-Catalyzed Synthesis of Sulfonyl Carbamates and Sulfonyl Ureas and Synthesis of Sulfonamides (Paper II) .......................................................................... 42 

2.2.1 Background and Aim .................................................................. 42 2.2.2 Existing Synthetic Protocols and Method Development ............ 43 2.2.3 Sulfonamides, Sulfonyl Ureas and Sulfonyl Carbamates- Scope and Limitations ......................................................................... 47 

2.2.4 Thermal and Chemical Stability of Sulfonyl-Containing Compounds .......................................................................................... 49 2.2.5 Mechanistic Studies .................................................................... 51 2.2.6 Summary (Paper II) ................................................................... 51 

3 PET Imaging Studies Focusing on the AT2R ............................................. 53 3.1 Synthesis of 11C-labelled Sulfonyl Carbamates via a Multicomponent Reaction Employing Sulfonyl Azides, Alcohols and [11C]CO (Paper III) ................................................................................. 53 

3.1.1 Background and Aim .................................................................. 53 3.1.2 Radiochemistry Method Development ....................................... 53 3.1.3 Model 11C-Carbamates ............................................................... 56 3.1.4 Precursor Synthesis ..................................................................... 57 3.1.5 Preparation of [11C]C21 .............................................................. 58 3.1.6 Prostate Studies ........................................................................... 59 3.1.7 Biodistribution and Small Animal Imaging ................................ 59 3.1.8 Summary (Paper III) ................................................................. 61 

3.2 Synthesis and Evaluation of a 11C-labelled Angiotensin II AT2 Receptor Ligand (Paper IV) .................................................................... 62 

3.2.1 Background and Aim .................................................................. 62 3.2.2 Precursor Synthesis ..................................................................... 62 3.2.3 Radiochemistry Method Development and Tracer Preparation .. 63 3.2.4 Autoradiography ......................................................................... 65 3.2.5 Biodistribution and Small-Animal Imaging ............................... 66 3.2.6 Summary (Paper IV) ................................................................. 67 

4 Synthesis of Novel Nitrogen Heterocycles via Multicomponent Reactions ....................................................................................................... 68 

4.1 A Microwave-Assisted Multicomponent Synthesis of Substituted 3,4-Dihydroquinazolinones (Paper V) .................................................... 68 

4.1.1 Background and Aim .................................................................. 68 4.1.2 Initial Discovery and Optimisation of Reaction Parameters ....... 69 4.1.3 Reaction Scope and Further Synthetic Applications .................. 70 4.1.4 Exploring the Reaction Pathway................................................. 73 4.1.5 Summary and Future Outlook (Paper V) ................................... 73 

5 Concluding Remarks .................................................................................. 75 

7 Acknowledgments...................................................................................... 77 

7 References .................................................................................................. 79 

Abbreviations

Ac acetyl ACE angiotensin-converting enzyme AcOH acetic acid Ar aryl ARB angiotensin receptor blocker AT1R angiotensin II subtype 1 receptor AT2R angiotensin II subtype 2 receptor Bmax receptor density Bn benzyl Bu butyl Boc tert-butoxycarbonyl C21 N-butyloxycarbonyl-3-(4-imidazol-1-ylme-

thylphenyl)-5-isobutylthiophene-2-sulfonamide CBD conditions-based divergence CO carbon monoxide CO2 carbon dioxide Conv. conversion CNS central nervous system CT computed tomography DABSO 1,4-diazabicyclo[2.2.2]octane bis(sulfur dioxide) DBU 1,8-diazabicyclo[5.4.0]undec-7-ene d.c. decay-corrected DMA N, N-dimethylacetamide DMF N, N-dimethylformamide DMSO dimethylsulfoxide dppf 1,1′-ferrocenediyl-bis(diphenylphosphine) Equiv. equivalents (molar) Et ethyl EWG electron-withdrawing group GC/MS gas chromatography with mass spectrometry GPCR G protein-coupled receptor HIV human immunodeficiency virus HOMO highest occupied molecular orbital HPLC high-performance liquid chromatography iPr isopropyl iPrOH 2-propanol

Kd dissociation constant keV kiloelectron volt Ki inhibitory constant L ligand LUMO lowest unoccupied molecular orbital Me methyl MCR multicomponent reaction MRI magnetic resonance imaging MRS modular reaction sequences MW microwave m/z mass-to-charge ratio Nf nonafluorobutanesulfonyl (nonaflyl) NMR nuclear magnetic resonance PET positron emission tomography P-gp P-glycoprotein RAS renin-angiotensin system r.t. room temperature (ambient temperature) RCY radiochemical yield SN2 substitution nucleophilic, bimolecular SRR single reactant replacement SPE solid-phase extraction SUV standardised uptake value tBu tert-butyl Tf trifluorobutanesulfonyl (triflyl) THF tetrahydrofuran TLC thin-layer chromatography TMAO trimethylamine N-oxide Ts p-toluenesulfonyl (tosyl) UV ultraviolet Xantphos 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene

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1 Introduction

1.1 Overview and Thesis Context The overall aim of the work in this thesis is the preparation, via multicompo-nent synthesis, of 11C-labelled compounds for use in preclinical in vivo imag-ing and biodistribution studies. The thesis contains 4 peer-reviewed papers and one manuscript. The introductory section (Section 1) provides a brief out-line of method development in organic synthesis, followed by a discussion on multicomponent reactions (MCRs), with emphasis being placed on the aza-Henry, Mannich and carbonylation manifolds. This is followed by an intro-duction to sulfonyl azides, including a comparison to alkyl and aryl azides. Thereafter, tracer development in 11C chemistry is addressed, including a brief outline of current methods of producing and harnessing carbon-11 carbon monoxide ([11C]CO). The biological target of the thesis work, the angiotensin II receptor subtype 2 (AT2R), is also discussed in Section 1, and in Section 2, sulfonyl azides are further addressed vis-a-vis their preparation from (het-ero)arylsulfonamides via a late-stage diazotransfer protocol (Paper I). The use of sulfonyl azides as precursors in carbonylative chemistry is discussed in Section 2 (Paper II). In Section 3 of the thesis, focus rests upon 11C-carbonyl-ation reactions used to prepare radiotracers for imaging and biodistribution studies of the (AT2R). In Paper III, chemistry developed in Papers I and II is applied in the preparation of precursor molecules and analytical standards re-spectively, with the aim of developing a protocol for preparation of 11C-sul-fonyl carbamates via Rh(I)-mediated carbonylation. This methodology was applied in the 11C-labelling of the first non-peptide AT2R agonist. In Paper IV, Pd(0)-mediated 11C-carbonylation with the aim of producing a 11C-la-belled AT2R ligand is discussed. Finally, in Section 4, multicomponent aza-Henry and Mannich reactions are used to synthesise a broad scope of novel nitrogen heterocycles, described in Paper V.

This thesis illustrates the collaborative and inter-disciplinary nature of pre-clinical research. A biological question (imaging of an endogenous receptor in healthy and diseased states) is posed, leading to the development of novel chemical tools. In addition to being used to address the original biological question, these tools will ideally be applicable to other purposes.

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1.2 Introduction to Method Development in Organic Synthesis The high level of complexity possessed by many organic compounds has fuelled the demand for development of methods of synthesising specific com-pound classes, as well as for carrying out specific transformations, e.g. the stereoselective addition of a carbon nucleophile to a prochiral molecule. Typ-ically, a multistep synthesis will contain a number of different challenges, each of which can be unique to the specific target compound. However, this situation also presents an opportunity for the organic chemist to explore a spe-cific transformation and increase its synthetic utility beyond the scope of the reaction being studied. A method may already exist for a similar transfor-mation, as confirmed by literature and database searches. However, most in-termediates are unique compounds and the input of methodology researchers will be required to improve existing methods. In a favourable research setting, serendipity may also play a role in the discovery of novel chemical transfor-mations, and researchers are given time to pursue investigations of side-reac-tions and other anomalies encountered during routine work.

Having identified the nature of a transformation (if novel) or its limitations in the specific synthetic manifold (if adopted from the literature), methodolo-gists will then strive to optimise it, often selecting a model compound that exhibits the chemical characteristics of the true substrate while at the same time being relatively simple and inexpensive. A number of parameters will be systematically screened with regards to their impact on reaction yield, includ-ing (but not limited to) catalyst/ligand identity and concentration, solvent, temperature and reaction time. The reaction outcome can be judged in a num-ber of ways; preferably, the crude reaction mixture is analysed spectroscopi-cally and the yield determined based on comparison to an internal or external calibrant, but the product can also be isolated using techniques such as silica gel chromatography.

With a suitably optimised protocol in hand, the chemist will then proceed to explore the scope and limitations of the developed reaction protocol. Sub-strates will be varied with respect to electron density, steric properties and the presence of functional groups that could potentially interfere with a catalyst or display incompatibility with the developed reaction conditions. Finally, the synthesis of a known compound using the developed methodology will be demonstrated.

The work in this thesis adheres to the philosophy described above, and the workflow is described in detail in the relevant sections.

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1.3 Multicomponent Reactions A multi-component reaction (MCR) is defined by the reaction of three or more components to form a single product, containing essentially all of the atoms from the reactants.1 However, as the simultaneous collision of 3 or more reac-tants is unlikely, MCRs most likely proceed by a number of stepwise reactions leading to the desired product. Using easily prepared or commercially availa-ble starting materials, products can be selectively synthesized in a one-pot fashion with limited waste generation and ideally with high ease of purifica-tion. Via secondary transformations of MCR-derived products, large libraries of biologically active and structurally diverse molecules can be built up in relatively few steps.2

The earliest MCR (see Scheme 1) is most probably the Strecker aminoni-trile synthesis, first described in 1850,3 which was followed by the Hantzsch dihydropyridine synthesis of 1882.4 In 1921, Passerini reported5 a three-com-ponent synthesis of α-acyloxy carboxamides, and in 1959, arguably the most well-known MCR, the Ugi reaction, was disclosed.6 Despite their long history, only during the past 10–15 years has scientific interest reflected the enormous utility of these reactions.1

Scheme 1. Several well-known MCRs.

Four strategies1 exist for the synthesis of novel chemical entities via MCRs: (a) replacement of a single reactant in an MCR (single reactant replacement, SRR, see Figure 1); (b) employment of a modular sequence involving a reac-tive intermediate (modular reaction sequences, MRS); (c) modifying reaction conditions to provide multiple molecular scaffolds from the same reactants (conditions-based divergence, CBD); and (d) combining different MCRs (MCR2). Strategy (a) has been employed throughout this thesis in the synthe-sis of substituted 3,4-dihydroquinazolinones and various 11C-labelled car-bonyl derivatives. Due to their inherent simplicity and one-pot approach,

O

R HNH3 HCN

NH2

R CN+ +

NH2

R COOH

H+ Strecker's amino acid synthesis

O

CO2EtNH3

CHO+ +

HN

EtO2C

CO2Et

Hantzsch's dihydropyridinesynthesis

O

R1 OHR2CHO R3NC+ +

R1

O

O

R2HN

O

R3 Passerini reaction

R1CHO R3NC R4NH2R2COOH+ + + N

O

R2

R4

R1

O

NH

R3 Ugi reaction

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MCRs are well-suited for radiochemical applications with short-lived radio-nuclides, as was demonstrated in Papers III and IV. In these papers and in the synthesis of 3,4-dihydroquinazolinones (Paper V), the role played by each component was systematically analysed to gain insight to its contribution to the overall outcome of the reaction. Figure 1. Systematic replacement of each component in a known MCR.

1.3.1 The Mannich and aza-Henry Reactions- An Expedited Route to Polysubstituted 3,4-Dihydroquinazolinones

Some of the most fundamental carbon-carbon bond-forming processes in or-ganic chemistry utilise addition of C-H nucleophiles to C=X bonds (where X is O or N). Of these, the Mannich reaction7 is one of the more well-known and has therefore been the subject of intense study.8–10 A key element in Mannich reactions is the formation of a reactive imine intermediate, which can then undergo nucleophilic attack by a variety of nucleophiles, forming a new car-bon-carbon bond adjacent to a nitrogen atom (see Scheme 1). Primary amines (or ammonia) activate the carbonyl electrophile to form an electrophilic imine. This can then be attacked by the enol form of a carbon nucleophile, resulting in a Mannich base. An important development in this field is the use of chiral catalysts such as (S)-proline, which enables control over the stereochemical outcome of the reaction.11

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Scheme 2. The Mannich and aza-Henry reactions, showing formation of imine.

The aza-Henry (or nitro-Mannich) reaction involves addition of a nitronate nucleophile to an imine electrophile, and has been known for over a cen-tury.12,13 The nitroamine products formed in this reaction are versatile inter-mediates, containing vicinal nitrogen-containing moieties in differing oxida-tion states. With the advent of stereoselective protocols, a wide range of β-nitroamines has been reported using this reaction manifold.14,15

In this thesis, the aza-Henry and Mannich reactions have been utilised in the preparation of 3,4-dihydroquinazolinones (see Scheme 2). This scaffold is present in biologically active compounds used in the treatment of convulsive disorders,16 infectious diseases,17 autoimmune diseases,18 cardiovascular con-ditions19 and inflammatory diseases.20 Traditionally, this interesting class of compounds may be prepared by using Grignard reagents to effect a cyclization of 2-carboxamidobenzonitriles.21 Alternatively, a cyclization/reduction se-quence starting from 2-trichloroacetamidobenzophenones,19,22 the ring closure of 2-acyl or 2-cyanocarbamates,23 or reaction of o-acyl or o-amino anilines with a suitable carbonyl compound16,19,24 could be used. In Paper V, a highly reactive iminium ion intermediate generated from an o-formyl carbamate was used in conjunction with a suitable carbon nucleophile to form the dihydro-quinazolinone skeleton in moderate to excellent yields.

1.3.2 Palladium and Rhodium in Organic Synthesis This thesis contains three papers (II, III and IV) in which organometallic in-termediates were employed in carbonylation reactions. For this reason, a brief glance at the history of organometallic compounds in synthesis may be of in-terest to the reader.

Following Bunsen’s investigations25 of cacodyl (a derivative of the dime-thylarsenic group) in the early 1840s, alkylzinc compounds were prepared by Frankland26 later during the same decade. Subsequent preparations of non-

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transition metals covalently bonded to carbon were reported before 1870, leading onto the second period of organometallic chemistry (1870s–1950s). This period was dominated by names such as Grignard, Schlenk and Gilman (among others), who demonstrated the preparation of organic derivatives of most metals. This was followed by the study of the metal carbonyls27 during the 1930s, when the foundations of modern transition metal organometallic chemistry were laid. However, it was not until the unintentional preparation of ferrocene by Kealy and Paulson28 in 1951 that the chemical community as a whole showed interest in this esoteric subdiscipline. The ferrocene era was characterized by new methods of investigation which made possible the un-derstanding of the behaviour of organometallic compounds based on their structures. This marked the birth of the modern period of the field, with intense interest being directed towards the biological, catalytic and industrial applica-tions of organometallic compounds, especially those based on transition met-als. The reader may be interested to know that many organometallic com-pounds are highly toxic, and early research in this field was not for the faint of heart. Bunsen’s description25 of cacodyl provides some insight to the work-ing conditions of early chemists:

“…the smell of this body produces instantaneous tingling of the hands and feet, and even giddiness and insensibility...It is remarkable that when one is exposed to the smell of these compounds the tongue becomes covered with a black coating, even when no further evil effects are noticeable.”

Palladium (Pd) In 1802, during his chemical investigation of platina ore, Wollaston isolated and characterised palladium (atomic number 46), originally thought to be a platinum alloy.29 This rare, silvery-white metal is one of the platinum-group metals, and displays physical properties similar to the other members of this group (i.e. ruthenium, rhodium, osmium, iridium and platinum). However, since the invention of the Wacker process in 1959, in which ethylene is oxi-dised to acetaldehyde,30 palladium has distinguished itself as one of the fore-most transition metals used in organic synthesis. This is partly due to the nar-row energy gap between its two main* oxidation states, 0 and +2, making it an ideal catalyst for both oxidations and reductions. Secondly, its diminished ten-dency to undergo one-electron transfer reactions reduces the number of prod-uct-consuming side-reactions in which it can participate. Thirdly, palladium readily forms complexes with high d-electron counts, low oxidation states and of a relatively large size, thus increasing its affinity to π and σ donors, which favourably influences its chemoselectivity. Finally, due to its electronegativity (2.2 on the Pauling scale), Pd-C bonds are rendered relatively non-polar, in contrast to the strongly polarised bonds seen when using organolithium and

* The Pd(III) and Pd(IV) oxidation states are also known, and Pd(IV) could potentially offer access to complementary catalytic processes to those catalysed by Pd(0)/Pd(II).206,207

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organomagnesium reagents (Grignard reagents). For this reason, most types of polar bonds reactive to Grignard reagents react sluggishly with palladium, providing a set of chemical tools largely orthogonal to the chemistry offered by more traditional organometallic chemistry involving lithium and magne-sium.

Organic synthesis as a discipline has benefited greatly from the contribu-tion of pioneers in the field of organopalladium chemistry, and the 2010 Nobel Prize in Chemistry was awarded to Negishi, Suzuki and Heck for their re-search into carbon-carbon bond-forming reactions catalysed by palladium.31–

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Rhodium (Rh) Like palladium, rhodium (atomic number 45) is also a late transition metal in the platinum group. Rhodium was discovered by Wollaston soon after his dis-covery of palladium (vide supra), and exhibits many of the characteristics as-sociated with the platinum group of metals. However, rhodium is the rarest of the non-radioactive elements, and its annual production is measured in kilo-grams, not tonnes. The most important application of rhodium is in the auto-motive industry, with approximately 80% of the annual global production used in catalytic converters. As world supply is dominated by just three coun-tries (South Africa, Russia and Zimbabwe), recycling remains an important source of rhodium.34

Organorhodium complexes are most commonly found in oxidation states of +1 and +3, but can, like other platinum-group metals, exhibit reversible oxidation states, thus enabling the catalysis of a broad range of transfor-mations.35 Hydroformylation, in which carbon monoxide (CO) is added to an alkene, is arguably the most important example of homogeneous catalysis, with an annual production in excess of 10 million tonnes.36 In the laboratory setting, the most common class of reaction in which organorhodium catalysts are used is asymmetric hydrogenation, whereby two atoms of hydrogen are added to one of two faces of an unsaturated substrate molecule.37 The im-portance of rhodium in this process was acknowledged by Knowles’ share of the 2001 Nobel Prize in Chemistry, in recognition of his work on chiral phos-phine/rhodium complexes.38

1.3.3 Carbonylation Reactions In this thesis, the term carbonylation is applied to any reaction in which CO (carbon monoxide) is catalytically introduced into an organic molecule.39† The term was coined by Walter Reppe in the 1930s, during his work in the field of

†Free radical-mediated and non-catalytic metal-mediated carbonylations extend beyond the scope of this thesis, as do Koch-like carbonylations utilising an excess of CO under acidic con-ditions for formation of carboxylic acids from alkenes.

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ethylene chemistry at BASF (Badische Anilin- und Soda-Fabrik).40 However, three-component reactions (such as the ones carried out in this thesis work) between a substrate containing a halide or pseudohalide, CO and a nucleophile were first described by Heck and co-workers in 1974. In their seminal papers, aryl and vinyl iodides were reacted with a palladium pre-catalyst in the pres-ence of aliphatic alcohols or amines, resulting in the formation of esters and amides, respectively.41,42 This approach represented a radical improvement to the “traditional” route, which relied upon the formation of a Grignard reagent that would react with CO2 to form a carboxylic acid that could be esterified or amidated. Many common functional groups (such as carbonyls and nitriles) cannot withstand the harshly basic and reducing conditions induced by Gri-gnard reagents, and in contrast, many Pd-catalysed reactions proceed under mild conditions at ambient or slightly elevated temperatures.

CO, despite its inherent toxicity and flammable nature, is a very versatile one-carbon building block that can be incorporated into organic molecules without generating undesirable side-products. Although it is (relatively) ther-mally stable, it is chemically reactive, especially towards Pd and other transi-tion metals and their corresponding organometallic compounds. Its reactivity stems from its ability to provide a non-bonding pair of electrons and an empty valence shell orbital, both centred on carbon. This makes it very similar to singlet carbene, in that it contains a Lewis-basic HOMO and a Lewis-acidic LUMO, increasing interaction (through pi backbonding) with the d orbitals of transition metals and strengthening the metal-carbon bond.

In this thesis, palladium and rhodium have both been employed in car-bonylative processes, starting from sulfonyl azides or aryl halides (Papers II, III and IV). Carbonylation reactions can be viewed as MCRs, as essentially all atoms from the starting materials are present in the final product and alter-ation or replacement of each component is possible. Scheme 3a depicts the formation of a carbonylated product from a suitable (pseudo)halide, CO and a nucleophile, and in Scheme 3b, the formation of sulfonyl carbamates and ureas from a sulfonyl azide, CO and a nucleophile is shown. As can be seen below, the carbonylation of a halide/pseduohalide substrate can be divided into 5 fundamental processes. Following conversion of the precatalyst (palla-dium with ligands) to an active 14-electron complex, palladium inserts itself between the π substrate and the leaving group (oxidative addition), forming a square-planar aryl-palladium (II) complex. The rate at which oxidative addi-tion occurs increases when the aryl group is electron-deficient, and electron-donating ligands on palladium further enhance its reactivity. At this point, CO attacks palladium, displacing a ligand, after which 1,1-insertion of CO to the R-Pd bond provides a site for nucleophilic attack. Following deprotonation by base, the metal centre is reduced by two electrons and product is formed (re-ductive elimination).43

21

Scheme 3a. Catalytic cycle of aminocarbonylation.

Scheme 3b. Carbonylation of sulfonyl azides, adapted from Paper II.

The carbonylation of arylsulfonyl azides (Paper II) presumably follows a similar pathway (Scheme 3b). The active catalytic species will generate a palladium-nitrene complex that is susceptible to attack by CO.44,45 Rearrange-ment of the acylpalladium complex and reductive elimination generates a highly reactive sulfonyl isocyanate, which can then be trapped by a suitable nucleophile, such as an amine or alcohol (see Paper II). However, the exact mechanistic pathway has not been fully elucidated, and Doi and co-workers have suggested that the reaction may proceed via a triangular Rh-nitrene-CO complex that loses Rh upon nucleophilic attack by aniline or alkoxide to give the corresponding urea or carbamate.46

22

1.4 Sulfonyl Azides-Structure and Synthetic Applications

The sulfonyl azide moiety (see Scheme 4) possesses a unique ability to act as a one-, two- or three-nitrogen source. Although certain physiochemical char-acteristics are shared by the entire azide family, the influence of the highly electron-withdrawing sulfonyl group causes the reactivity of sulfonyl azides to differ somewhat from that of alkyl and aryl azides. Scheme 4A shows a comparison of different classes of organic azides, with regards to bond lengths (and angles for arylsulfonyl azides). All organic azides share similarities in terms of N1-N2 bond lengths (average length of 1.258 Å) and non-linearity (170°, N1-N2-N3, where N3 is the terminal nitrogen), as compared to the lin-ear azide anion. However, studies on decomposition‡ temperatures of different classes of azides show that sulfonyl azides, while able to act as a complete azide unit, are also more susceptible to decomposition to the corresponding nitrene.47–49 This is due to the reduced double-bond character of the N1-N2 bond as a result of electron delocalisation onto the sulfonyl group.

Scheme 4B shows resonance structures for aryl/alkyl azides and sulfonyl azides. Structures 1c and 2c explain the tendency of all azides to decompose to dinitrogen and nitrene, but the influence of the adjacent electron-withdraw-ing group results in an increased tendency of sulfonyl azides to undergo N1-N2 cleavage.

The dipolar nature and behaviour of azides as a class towards nucleophiles (attack on N3) and electrophiles (attack from N1) can also be explained by the structures below.

‡ All azides are energy-rich molecules with the potential to display explosive properties. Violent decomposition reactions may be expected when the (C+O)/N ratio is <3, but this is a general rule and all azides should therefore be handled with appropriate caution.

23

Scheme 4. A, comparison of bond lengths and angles for different azide classes, and B, resonance structures of aryl/alkyl azides and sulfonyl azides.48,49

The chief area of use of sulfonyl azides in organic synthesis is in the prepara-tion of diazo compounds from activated methylene compounds (diazotrans-fer), first demonstrated by Curtius in 1926.50 However, the unique reactivity of sulfonyl azides has led to ever-increasing use of this interesting functional group during the past 20 years, in applications extending beyond diazotransfer and cycloaddition reactions.51–53 Scheme 5 depicts the use of sulfonyl azides as zero-,54–56 one-,57–59 two-60,61 and three-nitrogen53,62 donors in a number of representative reactions. The wide range of substrates and structural diversity of pendant functional groups bear witness to the utility of sulfonyl azides as both reagents and precursor molecules in organic synthesis.

(hetero)arylsulfonyl azides

NN

SNO O

(Het)Ar

1.696 Å

1.238 Å

1.113 Å

NN

Csp3N

1.288 Å

1.127 Å

NN

Csp2N

1.249 Å

1.116 Å

N N NNa

azide anion

1.166 Å

1.166 Å

NN

SNO O

(Het)Ar

168.0-174.2°

112.2-113.7°

R N N N R N N N R N N N R N N N

1a 1b 1c 1d

A

B

RS

O O

NN

N RS

O O

NN

N2a 2b 2c 2d

RS

O O

NN

N

RS

O O

NN

N

aryl and alkyl azides

24

Scheme 5. Sulfonyl azides as zero-, one-, two- or three-nitrogen donors.

In this thesis, a crystalline and shelf-stable diazotransfer reagent was used to prepare a number of sulfonyl azides from the corresponding sulfonamides (Pa-per I). In Paper II, these compounds were then used in palladium-catalysed carbonylation reactions, giving the corresponding sulfonyl ureas (amine nu-cleophiles) and sulfonyl carbamates (alcohol nucleophiles). In addition, sul-fonyl azides were used in direct displacement reactions, whereby substituted sulfonamides were prepared under mild reaction conditions. Using sulfonyl azide precursors prepared following the methodology in Paper I, Rh(I)-medi-ated carbonylation was used to prepare the corresponding 11C-labelled sul-fonyl carbamates in a multicomponent carbonylation reaction (Paper III).

25

1.5 Tracer Development in 11C Radiochemistry A tracer is a compound or atom that can be used to follow and quantitatively study a process in a living system without influencing the rate or outcome of the studied process. The fundamental idea behind this approach, first intro-duced by de Hevesy,63 is that all isotopes of a given element will interact sim-ilarly with a biological target. The chief benefit of using tracers lies in their ability to provide information about dynamic processes, at exceedingly low concentrations.64

1.5.1 Positron Emission Tomography (PET) In 1928, the existence of a subatomic particle equal in mass but opposite in charge to the electron was postulated by Dirac.65 In 1932, Anderson, during studies on particles produced in the upper atmosphere, identified a particle with the mass-to-charge ratio of an electron carrying a positive charge, which he dubbed “the positive electron”.66 The name “positron” was suggested dur-ing peer review of the manuscript in which he described his findings, and later on, both he and Dirac were awarded the Nobel Prize in Physics for their con-tributions (in 1936 and 1933, respectively). Other major advances in the field were the production of artificial isotopes by the Joilot-Curies (Nobel Prize in Chemistry in 1935), Lawrence’s invention of the cyclotron (Nobel Prize in Physics in 1939) and de Hevesy’s pioneering work using radioactive salts as tracers (Nobel Prize in Chemistry in 1943).63,67,68 Building on these discover-ies, the first medical applications of the positron were demonstrated in 1951,69,70 and in 1973, the first human positron emission tomography (PET) imaging device was constructed at Washington University.71 During the fol-lowing decades, technological advances in widely diverse fields such as ma-terials science and information technology have contributed to the emergence of PET as the premier nuclear medicine imaging modality, thanks to its uniquely high sensitivity and ability to provide information on a molecular level.72,73

The fundamental principle underlying PET is the ability of proton-rich ra-dionuclides to undergo decay, and produce a positron (β+) and a neutrino (ν), thereby achieving stability. This is known as β+ decay, and results in the con-version of a proton to a neutron.74

p → n + β++ ν (Equation 1)

The positron will then embark upon a zigzag journey through the matter which surrounds it, imparting its energy to the orbital electrons of atoms along its path. As it reaches an almost complete standstill, it will combine with an elec-tron in the absorbing material. This particle-antiparticle combination results in an annihilation event, whereby the two particles are obliterated and two

26

gamma photons with 511 keV of energy are emitted in essentially opposite directions (180° ± 0.5°). These photons can be detected by a detector ring sur-rounding the research subject, and a three-dimensional image showing radio-activity concentration can then be constructed (see Figure 2 for a schematic description). The distance travelled by positrons after emission contributes to uncertainty as to the original position of the decaying nucleus, and the energy (and hence, average distance travelled before annihilation) of the emitted pos-itrons will vary depending upon the radionuclide from which they originated. Moreover, due to non-zero momentum of the positron, many photon pairs will not be emitted at strictly 180°. This, and deflection of the positron (scattering), will further degrade resolution, and these effects, together with the finite dis-tance travelled by the positron prior to annihilation, place a lower limit of res-olution that can be achieved using PET.75 Table 1 summarises the properties of some common positron-emitting nuclides used in medical imaging.

Table 1. Properties of some of the radionuclides used in clinical PET imaging and research. Data compiled from various sources.74–76

Nuclide Synthesis β+ emission (%)

t1/2 (min)

Eβ+

,max

(MeV) β+ range in H2O (mm)a

11C 14N(p,α)11C 99.9 20.3 0.97 1.1 18F 18O(p,n)18F 96.7 109.8 0.64 0.6 15O 14N(d,n)15O 99.9 2.03 1.74 2.5 13N 16O(p,α)13N 99.8 9.96 1.20 1.5

68Ga From 68Ge 88.9 68.3 1.90 2.9

aAverage distance.

However, in addition to advanced imaging equipment, the requirement for an on-site cyclotron for production of short-lived isotopes represents a significant barrier to the introduction of PET in many countries. Nevertheless, reductions in equipment prices, decentralised production of radiopharmaceuticals and growth in demand have contributed to an increase in viability of this imaging technique.77 Moreover, combined PET/computed tomography (PET/CT) and PET/magnetic resonance imaging (PET/MRI) technology couples the molec-ular imaging power of PET with high-quality anatomical information, increas-ing the amount of information that can be gleaned from a single examination.78 The majority of PET examinations are carried out in the clinical setting, using 18F-FDG (2-deoxy-2-[18F]fluoroglucose) to visualise altered metabolic pat-terns indicative of many tumour types.79 Additional areas of use are in neurol-ogy and cardiology, as well as clinical research and drug development using animal and human subjects.80,81

27

Figure 2. Schematic view of a PET scan. Adapted from Wikimedia Commons.

1.5.2 Considerations in Tracer Development Using 11C

Radiochemistry The use of PET allows non-invasive visualization of biological processes at the molecular or cellular level.82–84 This is achieved through the incorporation of a short-lived radionuclide such as 11C (t1/2 = 20.3 min) into an organic mol-ecule, which can then be administered as a tracer. Therefore, the need for rapid synthetic methods that allow the synthesis, purification and formulation of a given radiopharmaceutical on a nanomolar scale within minutes creates inter-esting synthetic challenges. Due to the short half-life of 11C, it is preferable to introduce the radionuclide in the final step of the synthetic route, thus avoiding product loss due to radioactive decay. Furthermore, isotopic dilution (the mix-ture of stable isotopes with radioactive carbon-11) must be taken into account as this leads to a reduction in specific activity (see below). In theory, all car-bon-containing materials used in target construction, transfer lines, gas traps, reagents and reaction vessels are potential sources of stable carbon isotopes. Figure 3 shows a typical workflow in the preparation and isolation of com-pounds in 11C-carbonylation reactions (see also Supporting Information, Pa-per III).

28

Figure 3. Generic 11C-carbonylation workflow.

Affinity The tendency of a drug to bind to a given target is governed by its affinity, expressed as the inverse equilibrium dissociation constant Kd. Ideally, this value should be in the subnanomolar to nanomolar range, to allow detection even when target expression is low, as is the case for most targets. A receptor density value (Bmax) value at least 10-fold higher than Kd is desirable, although exceptions have been reported.85

Specific activity Specific activity is defined as the radioactivity per total mass of all isotopic compounds, typically expressed as a molar amount (gigabecquerel/µmol). This criterion is of utmost importance in tracer development, especially when targeting low-density receptors. Moreover, a high specific activity reduces the risk of pharmacological and toxic effects from the tracer, as only a small total amount of ligand will be administered.86 The maximum specific activity the-oretically achievable using carbon-11 is 3.4 x 105 GBq/µmol (i.e. all carbon atoms as carbon-11), but in practice, reported values are significantly lower.87

Labelling position Because tracer metabolism is often unavoidable, it is of utmost importance to ensure that labelled metabolites do not accumulate in the site of interest. Polar metabolites are preferred, as they will either fail to cross lipophilic membranes or undergo rapid washout. When several labelling positions are available in a target molecule, the position giving rise to the most polar metabolite is often preferred.86,88

Permeability A balance must be achieved between sufficient lipophilicity (expressed as the logarithm of the distribution coefficient of analyte in octanol and physiologi-cal buffer, logD) to allow passage through a lipid membrane, while at the same time avoiding non-specific binding associated with increasing lipophilicity.86 In addition to lipophilicity, descriptors such as polar surface area, hydrogen bond donors and protonation constant of the most basic centre have been used

29

in a predictive model of CNS permeability, facilitating early screening of po-tential tracers.89 Moreover, the action of efflux mechanisms such as those dis-played by P-glycoproteins (P-gp) must be considered, as these can potentially change the concentration of labelled drug in the CNS.90 This is especially im-portant for tracer molecules, due to the low concentration of substance admin-istered.

Selectivity The use of a selective tracer will result in a lower degree of interaction with other binding sites such as other receptors, receptor subtypes or transporters. Imaging of a target with lower expression levels than potential confounders requires a much higher selectivity, and to avoid ambiguous results, a tracer should display at least 30-fold selectivity for the target.86 This thesis contains two papers in which radiochemical syntheses were per-formed (Paper III and Paper IV). After the identification of a suitable biolog-ical target (see Section 1.6), a number of tracer candidates were selected (fol-lowing the above criteria) and chemistry method development (cold and hot) commenced. With a suitable radiochemical synthesis in place, in vitro screen-ing using autoradiography and cell-based assays was performed. This was fol-lowed by small animal imaging and biodistribution studies.

Purification and analysis of radiopharmaceuticals Due to the extremely small amounts of material synthesised in the field of radiochemistry, analytical techniques such as NMR cannot be applied for characterisation. Instead, HPLC with UV and gamma-count detectors is used to confirm radiochemical and chemical purity. In a typical experiment, an al-iquot of crude product will be mixed with an isotopically unmodified refer-ence compound (i.e. chemically identical with the exception of the radionu-clide which has been introduced). The UV detector will identify the isotopi-cally unmodified reference compound, and the gamma detector will confirm the presence of the labelled analyte (see Paper III, Supporting Information for a typical UV/gamma chromatogram). In addition to this, techniques such as radio-TLC and radio-MS can be used to provide further confirmation of analyte identity. In parallel with monitoring of the composition of the crude reaction mixture, a purification step using semipreparative HPLC and/or solid-phase extraction (SPE) will be performed. Radiochemical purity (the fraction of total radioactivity belonging to the compound of interest) exceed-ing 95% for biological examinations is desirable, and for non-terminal inves-tigations, the product solution must be pyrogen-free (in addition to formula-tion in buffer solution at physiological pH).91 Moreover, precursors and iso-lated radiopharmaceuticals must display some level of resistance to the radio-active environment in which they exist, and the use of additives such as ethanol or ascorbic acid to mitigate the effects of radiolytic decomposition is well-established.92

30

1.5.3 Carbonylative Reactions in 11C Radiochemistry The vast majority of 11C-labelling reactions utilise methylating agents such as [11C]CH3I93 or [11C]CH3OTf94 as electrophiles, resulting in a final molecule with a pendant methyl group. Although these protocols are well-established and amenable to automation, they require the target molecule to contain a me-thyl group, thus limiting the scope of suitable molecules. To address this issue, there has been an increasing interest during the latest decade to incorporate other one-carbon building blocks, of which 11C-labelled carbon monoxide ([11C]CO) is particularly attractive due to the large variety of biologically in-teresting compounds containing a carbonyl group and its availability through reduction of cyclotron-produced [11C]CO2. Synthesis of [11C]CO is techni-cally uncomplicated, requiring a single-step reduction of cyclotron-produced [11C]CO2 over a heated bed of molybdenum95 at 850 °C or zinc96 at 400 °C, and recently, two groups disclosed methods for homogenous chemical reduc-tion of [11C]CO2 using silanes.97,98

Although prior method development using isotopically unmodified CO has been of great benefit when working with [11C]CO, standard protocols typically rely on a large excess of CO at high pressure, due to its poor solubility in organic solvents.99 To circumvent this problem, two main approaches have been adopted. Either [11C]CO is forced into solution in a pressurised micro-autoclave, or chemical trapping reagents are used.100–105 In contrast, work by Eriksson et al. in 2012 showed that [11C]CO could be confined in small-vol-ume disposable reaction vessels at low pressure using soluble xenon as trans-fer gas (see Figure 4).106 This greatly simplifies the workflow and removes sources of contamination (from residual catalyst/reagents) in injection loops and autoclaves. The labelling chemistry in this thesis was carried out using both high-pressure equipment and low-pressure xenon transfer, with [11C]CO as the labelling precursor.

Figure 4. Schematic of low-pressure system in Xe transfer mode. Reproduced with permission.106

31

Pd-mediated Carbonylation using [11C]CO The majority of carbonylation reactions involving a nucleophile, electrophile and [11C]CO are palladium-mediated107 (the extremely small amount, approx.. 10–100 nmol of [11C]CO available for reaction means that the metal will most likely only react once). Despite an extensive body of research demonstrating the utility of CO, 11C-carbonylation reactions are relatively rare world-wide.84,99 In an effort to address this issue and demonstrate the viability of [11C]CO as a labelling precursor, Andersen and co-workers reported the syn-thesis and characterisation of pre-formed oxidative addition complexes which were then used to prepare several complex tracer molecules.108

In this thesis, Pd(PPh3)4 was used to label a number of aryl halide precursor molecules with [11C]CO in an aminocarbonylation reaction (see Section 3, Pa-per IV).

Rh-mediated Carbonylation using [11C]CO Although rhodium-catalysed carbonylation reactions are widely used in indus-try, their use in the synthesis of organic compounds is less widespread than for palladium.109 This is reflected by the relative scarcity in the literature of rhodium-mediated carbonylation reactions using [11C]CO, and of these reac-tions, the majority were carried out using a high-pressure autoclave.99 Not-withstanding this, rhodium has demonstrated its utility in speciality applica-tions with functional groups that cannot be labelled via palladium-mediated reactions.46,110

In this thesis, a number of 11C-labelled sulfonyl carbamates were prepared from the corresponding sulfonyl azides in a Rh(I)-mediated multicomponent reaction (see Section 3, Paper III).

32

1.6 The Renin-Angiotensin System as a Biological Target

1.6.1 The Angiotensin II Receptor Subtypes 1 and 2 (AT1R and AT2R)-Biology The renin-angiotensin system (see Figure 5) is responsible for maintaining cardiovascular homeostasis through its major effector peptide angiotensin II (Ang II). Renin is a proteolytic enzyme secreted in response to stimuli such as reduced renal perfusion pressure, and through its action upon angiotensin-ogen, a plasma globulin of hepatic origin, the decapeptide angiotensin I (Ang I) is released.111 Ang I in itself has no appreciable activity but after removal of the two terminal amino acids by the angiotensin-converting enzyme,§ angio-tensin II (Ang II), a potent vasoconstrictor, is formed.112 Ang II itself acts on two major receptor subtypes, the AT1 and AT2 receptors (AT1R and AT2R), discovered in 1989.113 Although both receptors are Class A (rhodopsin-like) G-protein coupled receptors (GPCRs), they only share 34% sequence homol-ogy and have different signalling pathways and physiological effects.114 Ac-tivation of the AT1R leads to effects such as vasoconstriction, salt and fluid retention and sympathetic activation, which lead to an increase in blood pres-sure.115,116

Figure 5. Simplified diagram of the renin-angiotensin system, showing drug classes and their targets. Organs not to scale.

§ The inspiration for angiotensin-converting enzyme inhibitors came from observing the effects of venom from the Brazilian pit viper Bothrops jararaca. This fact was heavily exploited during marketing of the first-in-class compound, captopril, to the trepidation of prescribers and patients alike.

33

As a result, several antihypertensive drugs that selectively block the AT1R are available on the market today (angiotensin-receptor blockers, ARBs).117,118 Conversely, stimulation of the AT2R leads to effects which counter those of the AT1R, most notably vasodilation and antiproliferation. Although the view that the negative effects of AT1R stimulation can be countered by activation of the AT2R is relatively widespread, this belief has been challenged.119 For instance, it has been shown that the function of the AT2R may become pro-hypertensive and pro-hypertrophic in the diseased state.111 For this reason, a careful analysis of the side-effects of AT2R stimulation during diseased con-ditions should be undertaken, due to the evidently complex pathophysiology involved.

Expression of the AT2R is widespread in the foetus but drops rapidly after birth, suggesting its importance in foetal development.120 In healthy adults, expression is very low and concentrated to the adrenals, uterus, ovaries, heart and certain areas of the brain.115,121 Interestingly, expression in adult tissue is upregulated during conditions such as myocardial infarction, brain ischaemia, stroke and renal failure and evidence for an anti-inflammatory and tissue-pro-tective role is supported by the findings of numerous studies.116,121–123 Moreo-ver, a recent study showing a decrease in expression of the AT2R in advanced prostate cancer suggests the possibility of a preventive role in disease progres-sion.124 For these reasons, selective AT2R agonists have been proposed as new therapeutic alternatives in the treatment of cardiovascular disease and prostate cancer.111

1.6.2 Nonpeptidic AT2R-Selective Compounds Early insights into functional diversity of Ang II receptors were gained using angiotensin peptide fragments and analogues in functional assays that meas-ured responses such as vascular contraction. Building on this work, research-ers at Du Pont discovered the first compounds selective for the AT2R in 1991.125 This was followed by a surge in interest in antagonists with equal affinities for both the AT1R and the AT2R, the idea being that a blockade would mimic the antihypertensive effects of ACE inhibitors. Four major com-pound classes materialised from these efforts, and a number of low nanomolar compounds were reported.126 Of these, a subset carrying a sulfonyl carbamate side-chain was found to include a nonpeptide AT1R agonist** (L-162,313),127 that served as a starting point for the synthesis of the first AT2R-selective ag-onists (see Figure 6 overleaf).128 A stepwise reduction of the northern imidaz-opyridine head group of L-162,313 resulted in a series of compounds with nanomolar affinity to the AT2R, and these were confirmed as agonists in a biological assay.129 The flagship compound of this series, M024/Compound

** At the time of its disclosure (1994), the functionality of L-162,313 at the AT2R was unknown, but in 2004 it was shown to be an agonist.128

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21 (hereafter designated C21) is currently in pre-clinical trials as a potential treatment for idiopathic pulmonary fibrosis,130 but it has also shown beneficial properties in models of cardiovascular disorders,131,132 stroke,133 prostate can-cer124 and spinal cord injury.134 In addition to its potential as a therapeutic tool, C21 has been heavily exploited as a chemical probe for investigation of AT2R function.122,135,136

Studies into the structure-activity relationship of nonpeptidic AT2R ago-nists revealed that the northern imidazole head group of C21 could be com-pletely replaced, with retained activity.137 These findings inspired the devel-opment of a series of benzamide-containing compounds138 with low nanomo-lar affinity for the AT2R (see Figure 6). Furthermore, the removal of the ben-zylic imidazole group reduces potentially detrimental inhibition of the cytochrome P450 system, lowering the risk of harmful drug-drug interac-tions.††139

In this thesis, selective AT2R agonists were used in PET imaging, autora-diography and biodistribution studies (Section 3, Papers III and IV).

Figure 6. Evolution of selective AT2R agonists from a balanced AT1R /AT2R ago-nist.

†† C21 is an inhibitor of CYP2C9 and CYP3A4.139

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1.7 Access to 11C-labelled AT2R Agonists- Labelling Strategies

Given the important role of the AT2R in disease states such as myocardial infarction, stroke and prostate cancer, an imaging agent capable of visualising receptor distribution and density in diseased and healthy states would be of great value in disease monitoring and assessment. For this reason, a need was identified for 11C-labelled AT2R agonists as PET imaging agents and tracers.

Figure 7. Potential labelling precursors (3–6) of 11C-labelled AT2R agonists.

A retrosynthetic analysis identified a number of potential options for the prep-aration of 11C-labelled AT2R agonists (see Figure 7). In the case of the ben-zamide analogues of C21, an aminocarbonylation approach was deemed via-ble, given the availability of methodology for incorporation of a radiolabel via the corresponding aryl halide (see Figure 7A).140,141

For C21 itself, several synthetic routes are feasible. Starting from [11C]for-maldehyde142 (available from [11C]MeI and trimethylamine oxide, TMAO), condensation with glyoxal, ammonium acetate and a suitable benzyl amine would furnish [11C]21 (Figure 7B). However, to the best of our knowledge, isotopic labelling of imidazole in the C2-position has not been demonstrated

S

SO O

NH

O

OBu

NH2

H11C

O

H

NH4OAc

O

OS

N

N

SO O

NH2

[11C]CO2

n-BuX

DBU

S

ZR

SO O

NH

OBu

O

*

NN

Z = CH2

S

I

SO O

NH

O

OBu

* = 11C

[11C]CO

HNR2

A

C

S

SO O

NH2

D

N

N

11C-phosgene/Se(0)

n-BuOH

[11C]CO

S

N

N

SO O

N3

[11C]CO

n-BuOH

E

Z = *CO =R =

(B-E)(A)

*

B

3

4 5 5

6

Benzamides, see Paper IV C21, see Paper III

N, N-dialky R

36

with a short-lived isotope. This route, although attractive due to the incorpo-ration of the radionuclide in a metabolically stable position, would require prolonged reaction times and multistep syntheses.‡‡ Alternatively, a trapping reagent such as DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) could be used to directly incorporate [11C]CO2 into the sulfonyl carbamate moiety of C21 (Fig-ure 7C).143 However, this elegant and operationally simple approach has not been demonstrated with sulfonamides as the nucleophilic component. Further-more, due to the low level of expression of the AT2R in healthy tissue, isotopic dilution of cyclotron-produced [11C]CO2 by atmospheric CO2 could poten-tially lead to decreased specific activity. A similar method, using selenium and [11C]CO for the preparation of 11C-labelled carbamates, circumvents these is-sues (Figure 7D).144,145 Alternatively, 11C-phosgene146 could be used to form a reactive intermediate that could be trapped by an alcohol, but this approach is technically non-trivial. Finally, Åberg and co-workers have successfully pre-pared 11C-labelled non-symmetric sulfonyl ureas using Rh(I), starting from the corresponding sulfonyl azide (Figure 7E).110 This approach would allow the incorporation of [11C]CO in one step, thus avoiding issues with prolonged reaction times, multistep reactions and possible isotopic dilution. It would, however, require a key sulfonyl azide precursor (6) and labelling chemistry for which novel methodology would have to be developed (see Section 2, Pa-per I and Section 3, Paper III).

With regards to the affinity and selectivity of C21, a low-nanomolar Ki value together with a very high selectivity for the AT2R provided a satisfactory starting point. However, using the predictive model89 of Zhang and co-work-ers, C21 possesses values for lipophilicity, polar surface area and molecular weight outside the most desirable range, indicating a reduced probability of CNS penetration. Given the fact that the blood-brain barrier can be compro-mised under certain disease conditions,147 the low predicted CNS penetrance of C21 may not present an obstacle to CNS imaging. Moreover, a lack of CNS penetration does not rule out the use of C21 as a tracer molecule for imaging of peripheral AT2R.

‡‡ 13C-labelled imidazoles have been prepared in modest yields, requiring elevated temperatures (200 °C) and 4 h of heating.208

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2 Sulfonyl Azides as a Versatile Entry Point to Novel Carbonylation Chemistry

2.1 Synthesis of Sulfonyl Azides via Diazotransfer using an Imidazole-1-Sulfonyl Azide Salt: Scope and 15N NMR Labelling Experiments (Paper I)

2.1.1 Background and Aim The sulfonyl azide moiety, as previously described, is a versatile functional group that can act either as a one or three-nitrogen precursor, or as a two-nitrogen donor to other molecules. However, “azidophobia”148 may have con-tributed to widespread reluctance to use azides in synthesis. Therefore, if a new azide synthesis protocol is to be adopted, certain conditions such as the use of environmentally benign solvents and availability of stable reagents must be met.

After selection of sulfonyl azide precursor 6 for use in a Rh(I)-mediated carbonylation reaction (see Section 1.7), a literature review was carried out to identify suitable methods to prepare sulfonyl azides. Traditionally, sulfonyl azides are prepared from the corresponding sulfonyl chloride (RSO2Cl) using sodium azide149 (NaN3), the most common precursor azide. However, this route is hampered by the chemical lability of sulfonyl chlorides, as well as the relatively limited number of commercially available compounds containing this moiety. Furthermore, solvent choice is of utmost importance, as NaN3 has been known to form explosive diazidomethane in the presence of halogenated solvents and exceedingly toxic and explosive hydrazoic acid in the presence of water.150 Therefore, primary sulfonamides (RSO2NH2) were selected as a starting point in sulfonyl azide synthesis, given their moisture and air stability and relative chemical inertness. The chief aim of this study was to develop methodology for the transformation of sulfonamides to sulfonyl azides, and apply said methodology in the preparation of structurally diverse sulfonyl az-ides.

38

2.1.2 Adaptation of Existing Protocols The study was initiated by identifying literature precedents for the transfor-mation of aryl sulfonamides to aryl sulfonyl azides. The first such transfor-mation was carried out by Raushel and co-workers in 2008, using trifluoro-methane sulfonyl azide (TfN3, 7, see Figure 8).151 This protocol circumvented the stability issues of sulfonyl chlorides and gave structurally diverse sulfonyl azides in high yields. However, 7 is costly, shock-sensitive and lacking a standardised synthetic protocol, must be prepared immediately prior to use. Nonafluorobutanesulfonyl azide (NfN3, 8), although also liquid at room tem-perature, is shelf-stable and efficient, as shown by Suárez and co-workers.152 Imidazole-1-sulfonyl azide153 hydrogen sulphate (9), is shelf-stable, crystal-line and can be made from inexpensive reagents. Importantly, it has been ex-tensively investigated154 in terms of shock-sensitivity and resistance to elec-trostatic discharge.§§ As a crystalline solid, its integrity can be confirmed by melting point determination, in addition to visual inspection and 1H NMR analysis.

Figure 8. Reagents used for preparation of sulfonyl azides from sulfonamides.

After 9 was synthesised following the literature procedure,154 a parameter screen was carried out with the aim of developing a simplified protocol (Table 2). Previous workers151,155 reported the use of a ternary solvent system, but with the final aim of developing a single or binary solvent protocol that did not require Cu additives or hydrocarbon solvents, the conditions of Goddard-Borger and Stick were selected as a starting point.153 p-Toluenesulfonamide (TsNH2, 10a) was selected as the substrate, and reaction parameters for the synthesis of p-toluene sulfonyl azide (TsN3, 11a) were investigated with re-spect to solvent, base identity and stoichiometry, and catalyst (see Table 2). Starting with 2 equiv. NaHCO3 and 5% Cu(II) in methanol, only trace amounts of product could be detected by LC/MS (Table 2, entry 1). Switching to a stronger base gave similar results (entry 2), but after the addition of water, the yield improved dramatically, presumably due to increased solubility of 9 (en-try 3). Removing the Cu(II) additive had no appreciable effect on isolated yield (entry 4), and thereafter, all reactions were run under metal-free condi-tions. Increasing the amount of base to 4 equivalents gave an almost three-fold

§§ Initial test reactions with the hydrochloride salt were abandoned after testing by Fischer and co-workers showed it to be as shock-sensitive as RDX, a military explosive.154

39

improvement in yield (entry 5), as did the use of the more environmentally-benign iPrOH (90%, entry 6).

Table 2. Optimisation of reaction conditions for synthesis of 11aa

entry base, equiv. solvent yield (%)a 1b NaHCO3, 2 MeOH traces 2b K2CO3, 2 MeOH traces 3b K2CO3, 2 MeOH/H2Oc 32 4 K2CO3, 2 MeOH/H2Oc 29 5 K2CO3, 4 MeOH/H2Oc 86 6 K2CO3, 4 iPrOH/H2Oc 90

aIsolated yields. b5 mol% CuSO4 added. c1/1 mixture. 0.5 mmol sulfonamide, 1–2 mmol base, 0.5 mmol 9, solvent (5.6 mL). c1:1.

2.1.3 Preparation of Structurally Diverse Sulfonyl Azides The optimised metal-free conditions were then applied to a panel of aryl and heteroaryl sulfonamides, including three registered drug compounds (Table 3). Sulfonamides with electron-donating substituents performed very well, and the synthesis of 4-methoxybenzenesulfonyl azide (11b) was complete within 8 h. Halogen and electron-withdrawing substituents were easily accom-modated, as were sterically hindered substrates (11c–11f). Despite the pres-ence of a distal basic nitrogen, 5-isoquinoline sulfonamide was smoothly con-verted into the corresponding sulfonyl azide (11g), but when the basic centre was situated ortho to the sulfonamide moiety, a much lower yield was ob-tained (11h). The presence of an external alkyne did not affect formation of the sulfonyl azide, and no N-sulfonyl triazole53 could be detected by LC/MS (11i). In order to evaluate the efficiency of this protocol in late-stage azide introduction and increase its attractiveness to medicinal chemists, three drug compounds were then tested using the developed conditions. Hydrochlorothi-azide,112 a potent diuretic acting on the distal tube, returned a yield of 76% (11j). Furosemide,112 a loop diuretic,*** was transformed into the correspond-ing sulfonyl azide in an excellent yield of 82% (11k), but the atypical antipsy-chotic sulpiride,112 despite prolonged reaction times, gave an isolated yield of 20% (11l). Initial concerns that the tertiary nitrogen in sulpiride was coordi-nating to the sulfur atom of 9 were dispelled by a control reaction in which 2

*** In a phrase that conjures a rather uncomfortable picture, loop diuretics are often described as causing ”torrential urine flow”.

40

equiv. Et3N was added to a mixture containing 9 and 4-methoxybenzenesul-fonamide (10b), leading to full consumption of starting material within 4 h. Finally, an alkyl sulfonamide was tested, but only traces of compound could be detected by TLC and GC/MS analysis, highlighting the need for an sp2 carbon adjacent to the sulfonyl group (11m).††† This could be due to the for-mation of an equilibrium between product and a negatively charged species derived from the diazotransfer reagent, which could then attack the newly-formed azide. An ideal diazotransfer reagent in this case would carry a suffi-ciently electron-poor aromatic group to ensure that its pKa (after diazotrans-fer) will be significantly lower than that of the alkyl sulfonamide. For this reason, 11e (pKa 8.94, cf. 11.07 for 10m) was also investigated, but no product could be detected in the reaction mixture.

Table 3. Preparation of structurally diverse sulfonyl azidesa

aIsolated yields, 0.5 mmol scale unless otherwise stated. b8 h reaction time. c1 mmol scale. d0.1 mmol scale. eStirred for 48 h. fCompound 11e instead of 9.

2.1.4 Mechanistic Studies Following the successful demonstration of the suitability of 9 for the conver-sion of sulfonamides to sulfonyl azides, a mechanistic study using isotopi-cally-labelled compounds was then undertaken (see Scheme 6). Although mechanistic studies to understand the formation of azides from primary amines have been described,156 the formation of sulfonyl azides from sulfon-amides had not yet been explored.

††† Other alkylsulfonamides tested were n-butylsulfonamide and (1S)-10-camphorsulfonamide, none of which gave detectable product.

41

The first step appears to involve a deprotonation of the sulfonamide to gen-erate the active nucleophile, evidenced by the lack of product formation when a weak base was used. Thereafter, two pathways are available: the nucleophile can either attack the electrophilic sulfur centre (Path A), or the terminal nitro-gen N3 (Path B). Attack on sulfur will expel an azide anion, which can return to displace the tosylamide group, forming the final sulfonyl azide product. Alternatively, attack on the terminal azide nitrogen will generate a tetrazine intermediate, which can then collapse to the sulfonyl azide product. Path A cannot be ruled out the basis of retention or inversion of configuration, due to the achiral nature of the sulfonamide, and there are reagents that operate by azide transfer, such as 2,4,6-triisopropylbenzenesulfonyl azide.157 Therefore, 10a carrying an isotopic label (15N-l0a) was subjected to the optimised condi-tions from Table 2, resulting in the exclusive formation of a single product with m/z of +1, as compared to the isotopically unmodified compound 11a. The identity of this new compound was confirmed as p-toluene sulfonyl azide-1-15N (15N-l1a). The retention of the 15N label (confirmed by 15N NMR) con-firms that 9 supplies two nitrogens via diazotransfer rather than three nitrogens by azidation, in line with earlier reports,158 and after submission of Paper I for peer review, a paper by Pandiakumar and co-workers confirming that im-idazole-1-sulfonyl azide indeed is a diazotransfer reagent became available online.159

Scheme 6. Proposed mechanistic pathway for formation of 15N-11a, adapted from Paper I. a. 15N-NH4Cl, Et3N, MeCN/THF, 140 °C, 4 h, 63%. b. Conditions from Ta-ble 1, entry 6.

R S

O

O

NH

p-tol

O

O

S

HO

O

NSR

p-tol

SO O

NH N N N O

OS

RN3

-

p-tol S

O

O

N N N+ Np-tol S

O

O

N N

Confirmed by 15N NMR

and EI-MSNot observed

p-tol

SO O

NH2

K2CO3

H+

Path A

p-tol

S OO

Cl

15N-10a

N N N O

OS

R

Path B

15

15

N3

15

15 15HN S

O

O

R+

R =N

N

NNN RS

O

ONS

O

O

p-tolH

15

NNN RS

O

ONS

O

O

p-tol15

H

p-tol S

O

O

N N N15N-11a

15

H2, Pd/C

a b

15N-11a

42

2.1.5 Summary and Future Outlook (Paper I) A late-stage diazotransfer (or azidation) reagent was required for the prepara-tion of a key sulfonyl azide precursor to be used in 11C-labelling experiments. A review of the literature identified a crystalline, shelf-stable reagent (9) for which extensive safety data was available, and initial test reactions demon-strated the utility of this reagent for the preparation of sulfonyl azides from sulfonamides. Notably, 30 structurally diverse (hetero)arylsulfonyl azides (in-cluding three functionalised pharmaceuticals) were prepared in good to excel-lent yields, without the addition of Cu salts. Finally, a mechanistic investiga-tion using 15N-labelled TsN3 showed that this useful reaction proceeds via a diazotransfer mechanism. This metal-free, late stage diazotransfer protocol will be of use to medicinal chemists wishing to prepare azide derivatives of biologically active sulfonamides. However, one limitation of this methodol-ogy is its non-compatibility with alkyl sulfonamides, and at present, sulfonyl chlorides provide the sole starting point for the synthesis of alkyl sulfonyl az-ides.

2.2 Sulfonyl Azides as Precursors in Ligand-free Palladium-Catalyzed Synthesis of Sulfonyl Carbamates and Sulfonyl Ureas and Synthesis of Sulfonamides (Paper II)

2.2.1 Background and Aim After development of methodology for the preparation of a broad range of sulfonyl azides (prepared via diazotransfer from the corresponding sulfona-mides), these sulfonyl azides were envisioned as precursors in the carbonyla-tive synthesis of sulfonyl carbamates and sulfonyl ureas. Sulfonyl-containing moieties (sulfonyl ureas, sulfonyl carbamates and sulfonamides) are important from both a medicinal chemistry perspective and in organic synthesis. The sulfonyl carbamate moiety can be found in molecules with anti-cancer,160 her-bicidal,161 lipid-regulating,162 and antibacterial163 properties. Furthermore, it is present in a plethora of AT1R and AT2R ligands164 (see Section 1.6.2). Sul-fonyl carbamates are used as nitrogen nucleophiles in the Mitsonobu reac-tion,165 as protecting groups for alcohols,166 and as dehydrating reagents.167

The sulfonyl urea group forms the backbone of many antidiabetic drugs that act on the pancreas to release insulin, an effect first discovered by chance during WWII.168 In addition to their use in the management of diabetes, the three main therapeutic areas in which sulfonyl urea-containing compounds have been investigated are oncology,169 infectious diseases170 and inflamma-tion.171

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Sulfonamides were the first class of antibacterial agents to be used system-atically, and their introduction in 1932 ushered in the era of chemotherapy as a means of managing infectious disease.172,173 Although they have largely been superseded by more modern compounds, they are still used today in the man-agement of certain bacterial infections, and the sulfonamide moiety is present in a wide range of registered drugs, including diuretics, anticonvulsants, anti-psychotics, antivirals and analgesics.174 In organic synthesis, amines can be protected as sulfonamides via reaction with an appropriate sulfonyl chlo-ride,175 and primary sulfonamides are widely used in the formation of imines.176

With this in mind, the chief aim of the current study was to develop a car-bonylative synthesis of sulfonyl carbamates and sulfonyl ureas. These sul-fonyl carbamates could then be used as reference compounds for characteri-sation of isotopically modified sulfonyl carbamates in later studies.

2.2.2 Existing Synthetic Protocols and Method Development Sulfonyl carbamates are traditionally prepared via N-acylation of a primary sulfonamide with a chloroformate or anhydride.177 Sulfonyl ureas can be pre-pared in a similar fashion, with isocyanates,178 phenoxycarbamates179 or sul-fonyl carbamates180 as the electrophilic component. As for sulfonamides, their chief route of preparation is via condensation of amines with a sulfonyl chlo-ride, but they can also be synthesised from the corresponding sulfonic acids,181 thiols,182 or from Grignard reagents and DABSO.183 An arguably more diver-gent approach to the synthesis of the above three functional groups would be from the reaction of an appropriate nucleophile with a sulfonyl isocyanate184 (see Scheme 7). Although sulfonyl isocyanates generally react rapidly and cleanly, their highly reactive nature makes storage and handling problematic, and consequently, the number of commercially available compounds is lim-ited. The typical method of preparation of sulfonyl isocyanates is from the reaction of sulfonyl ureas or sulfonamides with phosgene185 or oxalyl chlo-ride186 in dichlorobenzene (see Scheme 5). More recently, Ren and Jiao re-ported an in situ method of isocyanate generation from aromatic azides and atmospheric pressure CO under palladium catalysis, and this work was chosen as the starting point in the synthesis of sulfonyl carbamates and sulfonyl ureas from azide-derived sulfonyl isocyanates. However, storage and handling of CO is non-trivial, as it is a colourless, odourless, highly toxic and flammable gas, and therefore, during the 15 years, significant effort has been devoted to method development in the field of alternative CO sources.187 In 2003, Wann-berg and Larhed reported188 the use of Mo(CO)6 as a solid CO precursor via chemical liberation using DBU, and in 2012, Nordeman and co-workers dis-closed a modified two-chamber vial system that allowed CO transfer from Mo(CO)6 to an adjoining chamber containing the reaction mixture (see Figure 9).189

44

Scheme 7. Generation of sulfonyl isocyanate,185,186, aryl isocyanate, 190 and use of sulfonyl azide as precursor in synthesis of sulfonamides,54,55 sulfonyl ureas110 and sulfonyl carbamates. Adapted from Paper II.

This simple yet elegant approach does not rely on costly equipment‡‡‡ or high CO pressures and allows the carbonylation of reduction-sensitive substrates that might otherwise react with Mo(CO)6.

‡‡‡ The modified two-chamber vial system is available at an approximate cost of EUR 40.

in situhydrolysis

NH2-R3

HO-R2 R2-COOCl

HO-R2H2N-Ar

Known methods of generatingsulfonyl isocyanate

Paper II

Method 1a:Phosgene

DMAP

Base

C6H5OCONHHet

Jiao's work

N

PdCl2

CO N NH

O

O

R1HO-R1

Method 1b:Oxalyl chloride

NN

CO

SO

NH2

O

R1S

O

N

O

R1 CO

SO

N

O

ClC

O

R1-SnR3

SO

NH2

O

R1

SO

NH

O

R1 O

O

R2

SO

NH2

O

R1

SO

NH

O

R1 NH

O

R3

SO

N

O

R1C

O SO

NH

O

R1 O

O

R2

SO

N

O

R1N

N

NHR2R3

H2N-R, COS

O

NH

O

R1 NH

O

R2

NHO

MeCN

SO

N

O

R1

O

Sulfonyl azides as sulfonamide, sulfonyl urea and sulfonyl carbamate precursors

SO

N

O

R1

R3

R2

THF

350 bar

R3R4NH

Base

SO

N

O

R1R3

R4PdCl2 CO

SO

NH

O

R1 NH

O

ArPaper II

45

Figure 9. Chemical liberation of CO(g) from solid Mo(CO)6 after addition of DBU in two-chamber reaction vial. CCO, CO-release chamber; Crxn, reaction mixture chamber.

The initial study commenced with the treatment of 11a with butylamine (2 equiv.) and 5 mol% PdCl2 in DMA with chemical release of CO from Mo(CO)6 (see Scheme 8).

Scheme 8. Unexpected formation of sulfonamide 13a from TsN3 and BuNH2.

Interestingly, the desired sulfonyl urea 12a could not be detected by ESI-MS analysis, and instead, sulfonamide 13a was isolated in 54% yield. It was hy-pothesised that formation of 13a had occurred via direct substitution, in anal-ogy with the formation of sulfonamides from sulfonyl chorides and amines. Although this transformation has previously been reported,54,55 only three ex-amples exist in the literature. Using the methodology developed in Paper I, this transformation would allow the late-stage preparation of substituted sul-fonamides via a sulfonyl azide intermediate. Subsequent method development was carried out, with a clear trend towards more polar solvents and reduced temperatures being observed, and after addition of triethylamine (Et3N), the yield of 13a greatly improved (see Table 4).

46

Table 4. Optimisation of reaction conditions for synthesis of 13a

Entry Temp (°C) Solvent Yield (%)a 1 ambient Toluene 28 2 ambient MeOH 31 3 ambient THF 35 4 75 DMA 50 5 ambient DMA 55 6b ambient DMA 84

aIsolated yields. b1 equiv. Et3N added.

Thereafter, attention was re-directed towards the multicomponent synthesis of compound 12a. The only previously-reported carbonylative synthesis of sul-fonyl ureas required highly specialised equipment and costly Rh(I) catalysts, and was carried out using [11C]CO as the carbonyl source.110 Following the discovery that aliphatic amines gave rise to direct substitution products, aro-matic amines were evaluated as nucleophiles. Reaction of 11a and 2 equiv. aniline in the presence of 5% PdCl2 in DMA at 75 °C for 20 h gave a good isolated yield of 73%, and reducing the amount of aniline (thereby simplifying purification) to 1 equiv. resulted in an improved isolated yield of 76%.

Finally, conditions for the synthesis of 14a from 11a using n-BuOH as the nucleophilic component were investigated (Table 5). The three-component as-sembly of CO, 2 equiv. n-BuOH and 11a proceeded smoothly at 75 °C, but reducing the amount of alcohol was detrimental to isolated yield (Table 5, entries 1 and 2). However, catalyst loading could be reduced to 2 mol % with-out negatively impacting yield, and these conditions were used to prepare a number of structurally diverse sulfonyl carbamates (Table 5, entry 3 and Table 6c, entries 14b–14h).

47

Table 5. Optimisation of reaction conditions for synthesis of 14a

Entry PdCl2 (mol %) BuOH (equiv.) Yield (%)a 1 5 2 82 2 5 1 45 3 2 2 80

aIsolated yields.

2.2.3 Sulfonamides, Sulfonyl Ureas and Sulfonyl Carbamates- Scope and Limitations

Table 6a. Preparation of structurally diverse sulfonyl ureasa

aIsolated yields. bRun at 40 °C. cRun at 50 °C.

Using the optimised conditions for the synthesis of sulfonamides, sulfonyl ureas and sulfonyl carbamates from sulfonyl azides, a broad panel of sulfonyl azides, anilines, alcohols and amines was investigated (Tables 6a–6c). Ani-lines carrying electron-withdrawing and electron-donating substituents in para position were well tolerated (Table 6a, 12b and 12c), and notably, the antitumour agent 12d could be prepared in 54% yield using this isocyanate-free methodology. Using o-methyl aniline as the nucleophile gave a moderate yield of 45% (12e), but modification of the azide component was well-toler-ated, with both electron-poor and electron-rich azides performing excellently and returning the corresponding sulfonyl ureas in excellent yields (12f and 12g).

Mo(CO)6/DBU, n-BuOH2% PdCl2

DMA, 20 h, 75 °C

11a

Me

SN3

Me

SNH

14a

OBu

OO O O O

SAr

O

O

N3 SAr

O

O

NH

Mo(CO)6/DBU, R'C6H4NH2

2% PdCl2

11a, 11b, 11d ONHR'

12b (87%)bMe

SO O

NH

NH

OOMe

Me

SO O

NH

NH

OBr

Me

SO O

NH

NH

O

12c (67%) 12d (54%)

Me

SO O

NH

NH

O

Ac

SO O

NH

NH

O

MeO

SO O

NH

NH

O

12e (45%) 12f (87%)c 12g (90%)c

Me

Cl

R' = -4OMe, -4Br,-4Cl, -2Me

12b-12gDMA, 20 h, 75 °C

48

Table 6b. Preparation of sulfonamides from sulfonyl azidesa

aIsolated yields. bRun at for 5 days with 4 equiv. amine and 6 equiv. Et3N. cRun at for 4 days with 4 equiv. amine and 6 equiv. Et3N.

Next, the catalyst-free synthesis of substituted sulfonamides from amines and arylsulfonyl azides was evaluated (see Table 6b). As expected, primary amines performed excellently, and with hexylamine as the nucleophile, a yield of 86% was obtained (13b). Using a cyclic secondary amine (piperidine), the corresponding substituted sulfonamide was prepared in 90% (13c), whereas an acyclic secondary amine (diethylamine) gave a reduced yield (13d), despite the addition of excess amine and Et3N and prolonged stirring. Propargylamine was converted into the corresponding sulfonamide in a fair yield of 58% (13e), despite the presence of a terminal alkyne, but when an amino acid was em-ployed as the nucleophilic component, only traces of product could be de-tected via ESI-MS (13f). Similarly, poorly nucleophilic aniline gave no prod-uct (13g). Introduction of an electron-withdrawing substituent to the azide component was then investigated (13h), and initial results indicated a low tol-erance for this adjustment. However, amid concerns that the sulfonyl azide precursor was undergoing degradation (see Section 2.2.4), a test reaction with-out Et3N was conducted, returning the sulfonamide product in an excellent yield of 93%. Finally, furosemide analogue 11k was subjected to the opti-mised reaction conditions, giving the N-butyl sulfonamide in a modest yield of 23% (13i).

49

Table 6c. Carbonylative synthesis of sulfonyl carbamatesa

aIsolated yields. bSingle-chamber experiment.

Given the absence in the literature of carbonylative syntheses of sulfonyl car-bamates, a number of aliphatic alcohols and sulfonyl azides were subjected to the optimised conditions (see Table 6c). Secondary and tertiary alcohols per-formed well, giving moderate to high yields (14b–14d). In the case of 14d, the electron-withdrawing trifluoromethyl moiety likely reduced the nucleo-philicity of the alcohol. Attempts to further simplify the protocol by testing a single-chamber reaction were unsuccessful, and the presence of an electron-withdrawing trifluoromethyl substituent on the alcohol was detrimental to iso-lated yield. With regards to the azide component, deviations from 11a gener-ally resulted in diminished yields, albeit with retained tolerance for electron-withdrawing, electron-donating and halogen substituents (14e–14g). Notably, an sp2 carbon adjacent to the sulfonyl group is not required in this protocol, and sulfonyl azide 11m was converted to the corresponding sulfonyl carba-mate in an excellent yield of 80% (14h).

2.2.4 Thermal and Chemical Stability of Sulfonyl-Containing Compounds Sulfonyl ureas have been shown to undergo acid-catalysed hydrolysis, and furthermore, can supply the proton needed to initiate this process.191 For this reason, Et3N was added to the eluent used during purification, ensuring that the product would be isolated as the triethylammonium salt. Several sulfonyl carbamates were also seen to undergo hydrolysis, and were therefore isolated as the triethylammonium salts. Indeed, the sulfonyl carbamate moiety is known to undergo slow hydrolysis to the corresponding primary sulfonamide in aqueous solution, thus necessitating its protection as a salt. Due to concerns

50

that the sulfonyl azide starting materials were degrading at elevated tempera-ture, a stability test using 1H NMR§§§ was conducted on a panel of sulfonyl azides and sulfonyl chlorides to determine their stability (see Figure 10 and NMR spectra in Figure 11). Heteroarylsulfonyl chlorides such as 2-pyrim-idinesulfonyl chloride are known to decompose with loss of SO2 to furnish the corresponding pyrimidine-2-chloride.192 Similarly, sulfonyl azides have been shown to undergo slow degradation to the corresponding sulfonamide in pH-adjusted solutions,193 but were stable in our hands at ambient temperature and 50 °C in CDCl3 for up to 18 h (after which the experiment was terminated). In contrast, p-nitrobenzenesulfonyl chloride underwent degradation at ambient temperature, and within 120 min a significant amount of impurity could be detected (Figure 11). Although the other sulfonyl chlorides retained their chemical integrity, this study demonstrates the viability of sulfonyl azides as precursors for substituted sulfonamides. Hence, a sulfonamide can be carried through a multistep synthetic sequence, converted to the corresponding sul-fonyl azide, and transaminated using the direct displacement methodology de-scribed above.

Figure 10. Sulfonyl azides and sulfonyl chlorides evaluated in stability assay

Figure 11. 1H NMR spectra showing stability of p-nitrobenzenesulfonyl azide and degradation of corresponding sulfonyl chloride at ambient temperature at different timepoints. A = 0 min, B = 60 min, C = 120 min.

§§§ NMR acquisition parameters: relaxation delay = 60 s, acquisition time = 5.126 s, number of scans = 1, spectral width = 6398 Hz.

51

2.2.5 Mechanistic Studies To determine the reaction pathway of the sulfonamide formation and car-bonylation reactions, 15N-11a was once again employed (see Scheme 9). Fol-lowing the optimised procedure from Table 4, 15N-11a was treated with bu-tylamine, giving sulfonamide product 13a in 71% yield. This compound lacked the characteristic M+1 m/z ratio expected upon incorporation of a 15N label, suggesting that the reaction proceeds via a SN2-like pathway, whereby the nucleophile attacks the S(VI) centre, displacing an N3

- unit. The reaction of amines and sulfonyl azides has previously been reported to result in diazo-transfer to the amine,153 and this deazidation protocol provides an orthogonal reaction manifold for the preparation of substituted sulfonamides.

In contrast, when 15N-11a was subjected to the alkyoxycarbonylation con-ditions of Table 5, a single 15N-labelled product was formed in 69% yield, confirmed by ESI-MS and 15N NMR to be 15N-14a. This indicated that the reaction proceeds via a nitrene intermediate, which could then either react via a Pd(0)/Pd(II) mechanism reminiscent of other amino- and alkoxycarbonyla-tion reactions,194 or an oxidative Pd(II)/Pd(0) cycle analogous to the formation of ureas and carbamates.195 The latter pathway would require an external oxi-dant to regenerate the Pd(II) catalyst, and would also give product using a sulfonamide as the nucleophile. A control reaction using TsNH2 as the nucle-ophile did not give any product, and as the reaction was known to work in the absence of an external oxidant, the second pathway could be ruled out. More-over, the successful use of Pd(PPh3)4, a Pd(0) catalyst, provided further evi-dence that the alkoxycarbonylation of sulfonyl azides operates via a Pd(0)/Pd(II) mechanism (see Scheme 3a, Section 1.3.3).

Scheme 9. Mechanistic investigation using 15N NMR labels to elucidate reaction pathway of sulfonamide and sulfonyl carbamate formation. * denotes position of 15N label.

2.2.6 Summary (Paper II) A carbonylation protocol was required for the preparation of sulfonyl carba-mates (and sulfonyl ureas) from sulfonyl azides. These were synthesised ac-cording to the optimised procedure of Paper I, and after parameter optimisa-tion, a carbonylative synthesis of sulfonyl carbamates or sulfonyl ureas was

52

used to prepare a total of 33 compounds. Furthermore, a protocol for the syn-thesis of substituted sulfonamides from amines and sulfonyl azides was devel-oped, allowing the late-stage functionalisation of azide-containing molecules. A mechanistic investigation revealed that this reaction follows an SN2-like re-action pathway, involving nucleophilic attack and direct displacement of azide anion, shown by the lack of a 15N label in the final product. In contrast, the carbonylative sulfonyl carbamate protocol was found to proceed via a nitrene extrusion/isocyanate formation mechanism, resulting in retention of a 15N la-bel on N1 from the azide moiety. In total, 53 compounds were synthesised in moderate to excellent yields, including a known antitumour compound and a functionalised loop diuretic.

53

3 PET Imaging Studies Focusing on the AT2R

3.1 Synthesis of 11C-labelled Sulfonyl Carbamates via a Multicomponent Reaction Employing Sulfonyl Azides, Alcohols and [11C]CO (Paper III)

3.1.1 Background and Aim The carbonylative synthesis of sulfonyl carbamates starting from sulfonyl az-ides was demonstrated in Paper II. This methodology demonstrated the fea-sibility of this approach and allowed the preparation of isotopically unmodi-fied reference compounds for use in characterisation studies. Given the wide range of disease conditions in which the AT2R plays a protective role, a 11C-labelled AT2R agonist would be of great potential value for imaging of disease progression and receptor distribution. Using developed methodology from Pa-per I to prepare key sulfonyl azide precursor 6, subsequent efforts could then be directed towards the development of a radiochemical protocol for the prep-aration of [11C]C21 , for use in small-animal imaging and biodistribution stud-ies. An optimal tracer would provide insight into receptor density and tissue distribution of the AT2R, and could potentially enable imaging of both healthy and diseased tissue. This would provide valuable insight into the degree of disease progression in pathological conditions such as prostate cancer, and in the case of myocardial infarction and stroke, the extent of tissue damage could be inferred via increase in receptor expression. The aim of this study was therefore to develop methodology to label the sulfonyl carbamate moiety with carbon-11, apply this methodology in the radiolabelling of C21 and thereafter evaluate the performance of [11C]C21 in both in vitro and in vivo models.

3.1.2 Radiochemistry Method Development As shown in Section 1.7, several potential synthetic routes to [11C]C21 are available. Although traces of [11C]C21 (6) were observed when compound 5 was allowed to react with selenium, n-BuOH and [11C]CO in the high-pressure system (see Figure 7D), many 11C-labelled side-products were also formed, and technical issues with the high-pressure system101 (arising from catalyst and reagent carry-over in sample loops and the microautoclave), led to adop-tion of the sulfonyl azide route shown in Figure 7E. Notwithstanding these

54

limitations, the use of molecular selenium represents an operationally simple entry point to 11C-labelled sulfonyl carbamates (and ureas) from sulfonamides, via [11C]carbonyl selenides.144,196

Table 7. Optimisation of reaction conditions for synthesis of [11C]14i

Entry [catalyst] (mM)

[ligand] (mM)

Additive (mM)

Conv. (%)b

RCY(%)a

1c PdCl2, 2 - - 68 50 2 Pd(PPh3)4, 3 - - 82 66 3 [Rh(COD)Cl]2,

0.4 Xantphos, 1.2 - 50 20d

4 [Rh(COD)Cl]2, 0.4

dppf, 1.2 - 92 56

5 [Rh(COD)Cl]2, 0.4

PPh3, 1.2 - 89e 79e

6 [Rh(COD)Cl]2,

0.4 PPh3, 2.4 - 94 88

(55)d

7 [Rh(COD)Cl]2, 0.4

PPh3, 2.4 A, 120 77 50

8 [Rh(COD)Cl]2, 0.4

PPh3, 2.4 B, 120 52 50

9 [Rh(COD)Cl]2, 0.4

PPh3, 2.4 H2O, 440 7 4

10 - - - 5 4 11f [Rh(COD)Cl]2,

0.4 PPh3, 2.4 - 14 6

aNon-isolated radiochemical yield, decay-corrected (see Paper III, supporting infor-mation). Unless otherwise stated, [n-BuOH] = 80 mM, [11n] = 40 mM. All reactions carried out in oven-dried pear-shaped reaction vials (1 mL) with PTFE crimp caps, under Ar atmosphere. bConversion, percentage of non-volatile activity remaining in the reaction solution after flushing with N2, see Figure S1 in Paper III, supporting information. c[n-BuOH] = 110 mM. dIsolated radiochemical yield, decay-corrected. eAverage of two runs. f24 h old catalyst solution, exposed to ambient light, kept un-der Ar atmosphere. A = picolinic acid. B = phenylacetylene. * denotes position of 11C label.

55

Using chemistry developed in Paper I and II, a number of sulfonyl azide precursors were prepared and converted to the corresponding isotopically un-modified sulfonyl carbamates, which could then be used as analytical stand-ards in a radiochemistry setting. In order to achieve control over reactant/rea-gent concentration, minimise waste and enable the rapid screening of varied reaction conditions, a modular workflow relying upon stock solutions of the reaction components was adopted.

With previous work on aryl azides46 and sulfonyl azides110 as entry points, a preliminary screening of reaction conditions (Table 7) was carried out for the multicomponent synthesis of 11C-sulfonyl carbamates, using thiophene-2-sulfonyl azide (11n) as a model substrate, due to its similarity to C21. Given the favourable results achieved by Ren and Jiao190 in the palladium-catalysed formation of carbamates from aryl azides and alcohols, PdCl2 was selected for initial catalyst screening. Indeed, the work of Paper II confirmed the utility of this inexpensive and widely-used reagent in the formation of sulfonyl car-bamates from sulfonyl azides, alcohols and CO. However, due to its limited solubility in solvents in which 11C-labelling reactions are normally conducted (DMF, DMSO and THF), preparation of stock solutions of PdCl2 was prob-lematic. Hence, it was used in concentrations an order of magnitude higher than other catalysts (see Table 7, entry 1), resulting in a non-isolated radio-chemical yield of 50%. A large number of 11C-labelled side-products was also formed, and the difficulties encountered when working with PdCl2, as well as its propensity to oxidise CO to CO2 in the presence of water197 made tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) a more attractive alter-native. This Pd(0) complex is a common choice for transition metal mediated 11C-carbonylation reactions,99,198 and gave an increased non-isolated radio-chemical yield (entry 2). Earlier work110 demonstrated the utility of Rh(I) in 11C-carbonylation reactions of sulfonyl azides, and three in-house ligands were screened together with this catalyst (see Figure 12). Xantphos gave an isolated radiochemical yield of 20% (entry 3), and changing to dppf gave an increased conversion of [11C]CO but also led to the formation of a large num-ber of 11C-labelled side-products (entry 4). Gratifyingly, the ubiquitous PPh3 displayed superior performance compared to the afore-mentioned ligands (en-try 5), and increasing the concentration of PPh3 to achieve a P/Rh ratio of 3:1 in order to form Wilkinson’s catalyst199 gave an excellent conversion of 94% and an isolated radiochemical yield of 55% (entry 6). These conditions were selected for future work, and inspired by the example set by Collins and Glo-rius, a robustness screen200 of the reaction manifold was carried out (entries 7–11). This would aid in determining the tolerance of the optimised reaction conditions to factors not normally considered in method development, such as external contaminants, ambient light and air (see Figure 12). Surprisingly, the presence of both a free carboxylic acid and a basic nitrogen centre that could potentially interfere with metal catalysts was tolerated (albeit with the for-

56

mation of several 11C-labelled side-products, entry 7). Similarly, a non-iso-lated radiochemical yield of 50% was observed using phenylacetylene as an additive (entry 8). This indicated that the sulfonyl azide starting material was available for reaction, despite its ability to undergo azide-alkyne cycloaddi-tion.148 Conversely, the addition of excess H2O proved to be detrimental, as did the omission of catalyst (entries 9 and 10). Finally, the use of 24 h old stock solutions exposed to light (but kept under Ar)**** resulted in a non-iso-lated radiochemical yield of 6% (entry 11) most probably due to catalyst de-composition. These findings emphasised the robustness of the reaction mani-fold to chemical contamination, but highlighted the need to maintain an oxy-gen- and water-free atmosphere.

Figure 12. Ligands and additives used in method development of 11C-labelling of sulfonyl carbamates.

3.1.3 Model 11C-Carbamates With optimised conditions in hand for the preparation of functionalised 11C-labelled sulfonyl carbamates, a number of alcohols and sulfonyl azides were evaluated (see Table 8). As demonstrated by the synthesis of [11C]14i, het-eroarylsulfonyl azides and primary alcohols were well-tolerated, as were sec-ondary alcohols, with [11C]14b being isolated in an excellent radiochemical yield of 57%. However, switching to a tertiary alcohol proved to be detri-mental to both conversion of [11C]CO and isolated radiochemical yield ([11C]14c). Thereafter, the effect of varying the sulfonyl azide component was evaluated. Electron-withdrawing substituents in para position were well-tol-erated, albeit with slightly reduced conversion and radiochemical yield ([11C]14e). The conversion of sulfonyl azide 11c to the corresponding 11C-sulfonyl carbamate proceeded smoothly ([11C]14f), but the introduction of an electron-donating methoxy substituent in para position led to a reduction in conversion and isolated radiochemical yield, presumably due to increased electron density on the sulfonyl moiety and thereby altered bond strength of the N1-N2 bond ([11C]14g). Gratifyingly, sulfonyl azide 11m (prepared from

**** Although all stock solutions were kept under a positive pressure of argon gas during solvent withdrawal, the possibility of air entering through the pierced PTFE crimp cap cannot be ruled out.

NOH

OP

Ph Ph

Ph

FeP

Ph

PhP

Ph

PhO

Me Me

P PPhPh PhPh phenyl-

acetylenepicolinic

acidtriphenyl-phosphinedppfXantphos

57

the corresponding sulfonyl chloride) gave the corresponding 11C-labelled sul-fonyl carbamate in a good yield of 51%, in agreement with the findings of Paper II. Taken together, these results showcase the success of the multicom-ponent approach in preparation of a small library of isotopically modified sul-fonyl carbamates. The use of stock solutions increases ease of reagent han-dling and is well-suited to the special working conditions of the radiochemis-try laboratory.

Table 8. Selected 11C-sulfonyl carbamatesa

aIsolated yields, decay-corrected, based on activity remaining after N2 purge (con-version, percentage of non-volatile products formed from [11C]CO). bNon-isolated radiochemical yield, decay-corrected. Reactant concentrations from Table 7, entry 6. * denotes position of 11C label.

3.1.4 Precursor Synthesis The synthesis of labelling precursor 6 largely followed the literature proce-dure.129,201 Starting from the commercially available tert-butyl thiophene sul-fonamide I, the isobutyl side-chain was introduced via lithiation and quench-ing with isobutyl bromide to give II. The alkylation reaction invariably led to a mixture of products, including dialkylated thiophene and N-alkyl thiophene, requiring painstaking purification by silica gel chromatography. N1-alkylation of imidazole with 4-bromobenzyl bromide provided III, which was coupled with boronic acid IV (prepared via lithiation/borylation of II) in a Suzuki-Miyaura32 cross-coupling reaction using Pd(PPh3)4 as the palladium source. The resulting 3-aryl thiophene (V) was treated with TFA, rendering free sul-fonamide 5. After isolation of sulfonamide 5, the optimised diazotransfer con-ditions from Paper I were then applied, giving key sulfonyl azide precursor 6. Isotopically unmodified C21 was prepared via N-acylation of 5 using n-

58

butyl chloroformate, providing an analytical standard for use in 11C-labelling experiments.

Interestingly, sulfonyl azide 6 could be used in 11C-labelling reactions di-rectly after aqueous work-up and drying over MgSO4, thus increasing the at-tractiveness of this approach.

Scheme 10. Preparation of labelling precursor 6. Reagents and conditions: (a) KOH, imidazole, DMF, reflux, 16 h; (b) i. n-BuLi, THF, -78 °C ii. 1-bromo-2-methylpro-pane, r.t. 16 h; (c) i. n-BuLi, THF, -78 °C ii. B(OiPr)3, r.t. 16 h; (d) III, Pd(PPh3)4, NaOH (aq.), toluene, reflux, 3 h; (e) TFA, 16 h; (f) 9, iPrOH/H2O, K2CO3, r.t., 20 h; (g) Butyl chloroformate, Et3N, r.t., 3 h.

3.1.5 Preparation of [11C]C21 As mentioned above, initial efforts to label C21 with carbon-11 using molec-ular selenium were foiled by the low solubility of selenium, and therefore the sulfonyl azide pathway was pursued. Using the developed methodology for 11C-labelling of sulfonyl carbamates in the carbonyl position, compound 6, n-BuOH and [11C]CO were heated at 100 °C for 5 minutes in THF. Generally, conversion of [11C]CO into non-volatiles proceeded smoothly, with an aver-age value of 66% being achieved. Interestingly, increasing the length of cy-clotron bombardment from 10 to 20 minutes was detrimental to both the de-gree of conversion and isolated radiochemical yield, suggesting that either the precursor or the product was unstable in conditions of high background radi-oactivity. Purification of the chromatographically challenging zwitterionic product was carried out using semi-preparative HPLC with a basic buffer, and

59

[11C]C21 was obtained in 24±10% decay-corrected radiochemical yield (ap-prox. 1 GBq after purification, average of 9 runs). With a robust synthesis of [11C]C21 in hand, biological evaluation could then be performed.

3.1.6 Prostate Studies As demonstrated by Guimond and co-workers, the RAS is an attractive poten-tial target in treatment of prostate cancer.124 The expression of AT2R was seen to diminish as a function of disease progression, and selective AT2R stimula-tion with C21 gave a decrease in non-tumoral cell number. This suggests that the AT2R plays a protective role against prostate cancer development. For this reason, it was envisioned that an AT2R selective 11C-labelled compound would be of value in monitoring of disease progression.

The DU145 cell line (a brain metastasis of human prostate carcinoma202) was chosen for evaluation of the in vitro binding characteristics of [11C]C21 . Although absolute levels of measured activity were low, measured activity was significantly lower (p = 0.03) in samples that were pre-incubated with non-labelled C21 (see Figure 13). This indicated the potential utility of [11C]C21 as a tracer for prostate imaging. However, as the expression of the AT2R is limited in most tissue, future studies should focus on cancer cell lines that express higher levels.

Figure 13. Specific binding of [11C]C21 to DU145 cells. For the pre-saturation of receptors, an excess of non-radioactive C21 was added. Data are presented as mean values from three samples±SD.

3.1.7 Biodistribution and Small Animal Imaging After demonstration of proof of concept, biodistribution and live-animal PET imaging studies commenced. Six healthy female rats were administered a dose of [11C]C21 through the tail vein, after which they were sacrificed at specific

60

timepoints (10, 20 and 40 minutes post-injection). Their organs were meas-ured for activity and standard uptake values were calculated (see Equation 2).

(Equation 2)

As seen in Figure 14, [11C]C21 accumulates predominantly in the excretory organs (liver and kidneys). No accumulation occurred in locations reported to contain AT2R-expressing cells, such as brain, pancreas and the reproductive organs.111 With regards to brain penetration, it is not known whether C21 is a P-gp substrate and therefore susceptible to active transport from the CNS. Given that expression of AT2R is upregulated in conditions such as brain is-chaemia,121 CNS penetration is a desirable property of any AT2R ligand.

Figure 14. Organ uptake of [11C]C21 in healthy female rats. BL – blood; HE – heart; LU – lungs; LI – liver; PA – pancreas; SP – spleen; AD – adrenals; KI – kidneys; INS- – small intestine without contents; INS+ – small intestine with contents; INL- – large intestine without contents; OV – ovaries; MU – muscle; BR – brain. Data are presented as mean values and error bars represent the SD.

For imaging purposes, two male rats were administered a bolus dose of [11C]C21 and scanned in a micro-PET-CT camera for 40 min. The results largely match those of the biodistribution ex vivo studies shown above. The only organ clearly visible is the liver, indicating rapid metabolism and non-specific binding (see Figure 15).

)(/)(

)/(

gTBWBqInjRA

gBqRASUV

61

Figure 15. PET-CT image, showing in vivo distribution of [11C]C21 in a healthy male rat. Grey scale (CT), 200–1500 Hounsfield units, colour scale (PET) SUV = 0 to 20 (black to red). White arrows show liver in 3 projections and kidneys in trans-axial projection.

3.1.8 Summary (Paper III) A new general and robust method for preparation of 11C-labelled sulfonyl car-bamates starting from the corresponding sulfonyl azide has been developed. This multicomponent protocol enables the rapid assembly of structurally di-verse sulfonyl carbamates and was demonstrated by the synthesis of 12 model 11C-labelled sulfonyl carbamates (see Table 8 and Paper III). The radiolabel-ling of a potent, non-peptide AT2R agonist (C21) was then successfully per-formed and the isolated compound was found to bind specifically in prostate tumour cells expressing AT2R. The biological evaluation of [11C]C21 indi-cates rapid metabolism and excretion and taken together, these results indicate that [11C]C21 is not a suitable PET tracer for imaging of AT2R receptor dis-tribution. However, given the potential utility of an AT2R-selective imaging agent in individualised management of prostate cancer therapy, as well as im-aging of other disease states, these results provide an entry point into the de-velopment of future tracers to meet this need.

62

3.2 Synthesis and Evaluation of a 11C-labelled Angiotensin II AT2 Receptor Ligand (Paper IV)

3.2.1 Background and Aim As mentioned in Section 1.6.2, a series of AT2R agonists carrying a benzamide head group instead of the northern imidazole was reported in 2008 (see Figure 6).138 These compounds are highly amenable to incorporation of a short-lived radioisotope such as carbon-11 via transition metal-mediated aminocarbonyl-ation.140 As shown in Paper III, a multicomponent approach consisting of a nucleophile, substrate and [11C]CO allows rapid access to small libraries of 11C-labelled carbonyl compounds. The alternative route to such compounds, via [11C]CO2 and the corresponding Grignard reagents, suffers from the draw-backs outlined in Section 1.3.3, such as low functional group tolerance to-wards the harshly basic Grignard conditions, and moisture-sensitivity of said Grignard reagents. Furthermore, the use of [11C]CO2 as a labelling tracer in-creases the risk of isotopic dilution by atmospheric carbon dioxide, thus low-ering specific activity. This is especially important when attempting to image a receptor with naturally low expression, such as the AT2R. In this vein, three compounds displaying low nanomolar affinity for the AT2R were selected as leads in tracer development (See Scheme 11, 15i–15k). As discussed in Sec-tion 3.1.1, the potential benefits of an AT2R-specific PET tracer in imaging of healthy and diseased tissue were the driving force behind this project.

3.2.2 Precursor Synthesis Synthesis of the benzamide precursors138 followed a similar route to that de-scribed in Paper III. Key intermediate IV was coupled with different aryl iodides in a Suzuki-Miyaura reaction.32 The resulting tert-butyl protected sul-fonamides (15a–15d) were deprotected with BCl3 and used without purifica-tion to give sulfonyl carbamates 15i–15k via N-acylation. In the case of 15e and 15f, the free sulfonamides were isolated for use as analytical standards in the radiochemistry laboratory. An alternative synthetic route to 15i via a one-pot aminocarbonylation protocol starting from aryl iodide 3 resulted in a com-plex mixture of products and was therefore abandoned (see Scheme 11).

63

Scheme 11. Synthesis of precursors and isotopically unmodified standards for radio-labelling of benzamide AT2R agonists. Reagents and conditions: (a) p-substituted aryl iodides, Pd(PPh3)4,Na2CO3, H2O, DME, EtOH, 10 min, 110 °C (MW); (b) BCl3, CHCl3, 1 h, r.t.; (c) n-butyl chloroformate, DCM, Et3N; (d) Pd(OAc)2, DBU, Mo(CO)6, diethylamine, THF, 1 h, 110 °C (MW).

3.2.3 Radiochemistry Method Development and Tracer Preparation As previously mentioned, compound 3 was chosen as the radiolabelling pre-cursor for all 11C-labelled benzamide AT2R agonists. A model three-compo-nent reaction system comprising 3, diethylamine and [11C]CO was evaluated with respect to concentration of reactant/catalyst concentration and tempera-ture. Pd(PPh3)4 was selected as the catalyst given its proven track record in similar applications,140 and all reactions were carried out using high-pressure concentration of [11C]CO in a micro-autoclave.101

The optimisation studies were initiated using an excess of 3, in accordance with earlier observations that an excess of aryl iodide was required to facilitate oxidative addition of palladium into the carbon-halogen bond (Table 9, entry 1).140 However, this approach resulted in a low non-isolated RCY and the rel-ative concentration of 3 was therefore reduced to match that of the catalyst. Indeed, sulfonyl-containing groups can act as nucleophiles under basic condi-tions, and an increase in the amount of amine nucleophile gave a concomitant increase in RCY (entry 2), whereas an excess of catalyst with respect to aryl halide concentration was detrimental (entry 3). An equimolar concentration of 3 and catalyst was beneficial (entry 4), but non-isolated radiochemical yield was seen to decrease with decreasing temperature (entries 5 and 6). During method development, a 11C-labelled side-product more polar than [11C]15i was observed on a number of occasions, possibly due to hydrolysis of the sul-fonyl carbamate moiety to give the free sulfonamide [11C]15f. However, the observed impurity did not co-elute with the analytical standard (15f), and the consistent failure of a two-step approach to synthesise [11C]15i via N-acylation of 11C-labelled sulfonamide [11C]15e prompted the use of 3 as the precursor in all future labelling experiments.

64

Table 9. Optimisation of reaction conditions for radiolabelling of 15i via Pd-medi-ated aminocarbonylation

Entry [cat] (mM)

[Et2NH] (mM)

[3] (mM)

Temp (°C)

Conv. (%)b

RCY (%)a

1 5 100 14 130 87 9 2 9 1000 9 130 93 34 3 9 500 7 110 89 16 4 9 500 9 110 92c 44c 5 9 500 9 100 93c 38c 6 9 500 9 70 93 10

aNon-isolated radiochemical yield, decay-corrected (see Paper III, supporting infor-mation). bConversion, percentage of non-volatile activity remaining in the reaction solution after flushing with N2.cAverage of two runs. * denotes position of 11C label.

The optimised conditions were then applied to the multicomponent synthesis of two other 11C-labelled AT2R agonists, displaying the versatility of [11C]CO in a library-based approach to different 11C-labelled carbonyl compounds (see Table 10).

Excellent conversion of [11C]CO into non-volatile products was seen in all three cases, with >1 GBq of product being isolated within 50 minutes of cy-clotron bombardment, and in the case of [11C]15j, a specific activity of 180 GBq/µmol was achieved. However, although the three compounds display similar selectivity to the AT2R, [11C]15j was selected for further studies on grounds of its lower lipophilicity (calculated), which reduced the risk of non-specific binding associated with higher lipophilicity.

65

Table 10. Synthesis of 11C-labelled AT2R agonists via multicomponent aminocar-bonylation

Entry clogD7.4

a Ki (nM)b

Conv. (%)

RCYc

(non-isolated %) RCYd

(isolated %) [11C]15i 5.94 3.0 92e 44e 9 [11C]15j 4.84 2.6 82 49 34 [11C]15k 5.79 1.0 76 58 16

aCalculated using Marvin Beans v.16.5.30.0. bAT2R, Ki AT1R >10000 nM. cSee Pa-per III, Supporting Information. dIsolated yields, decay-corrected, based on activity remaining after N2 purge. eAverage of two runs. * denotes position of 11C label.

3.2.4 Autoradiography With a suitable 11C-labelled AT2R agonist in hand, in vitro autoradiography using tissue slices from pig and rat was then conducted (see Figure 16). Spe-cific binding was observed in rat brain, pancreas and kidney, as well as pig adrenals. Interestingly, no binding at all was observed in rat liver.

66

Figure 16. Autoradiography of selected tissue sections using [11C]15j. Specific bind-ing confirmed by treatment of sections with 15j (2.5 µM). Adapted from Paper IV.

3.2.5 Biodistribution and Small-Animal Imaging Next, in/ex vivo studies (small animal PET and biodistribution) were con-ducted using male and female rats (Figures 17 and 18). A 40 minute dynamic PET scan revealed very high liver and bladder uptake of activity after injection with [11C]15j. The rapid accumulation of activity in the urinary bladder indi-cated a high rate of metabolism, and no specific binding was observed.

Figure 17. Dynamic PET scan of female rat injected with 12 MBq of [11C]15j. Whole body view. Adapted from Paper IV.

Bladder

Kidneys

Liver

Tail

67

The findings from the imaging study were confirmed by the biodistribution study. As can be seen in Figure 18, the majority of measured activity was in the liver, with a significant amount also being observed in the kidney and in-testines. This provided further confirmation that [11C]15j underwent rapid me-tabolism, thus precluding its use as a PET tracer. Furthermore, [11C]15j could not be detected in the sexual organs or brain, despite earlier reports116 showing the existence of AT2R in these organs.

Figure 18. Ex vivo biodistribution of [11C]15j. Animals were injected with 18 MBq [11C]15j and sacrificed after 40 min. Adapted from Paper IV.

3.2.6 Summary (Paper IV) Three high-affinity benzamide AT2R compounds were labelled with carbon-11 in a multicomponent aminocarbonylation reaction. Isolable yields were achieved in all three cases, and [11C]15j was prepared in 29% decay-corrected yield, with a specific activity of 180 GBq/µmol.

[11C]15j was evaluated with three pre-clinical methods (autoradiography, biodistribution and PET imaging), and specific binding was observed using autoradiography. However, small-animal imaging and ex vivo biodistribution showed rapid excretion of the radiotracer. Although the studied compounds were sufficiently selective and potent, future efforts in this field should be directed towards preparation of ligands with enhanced metabolic stability and reduced lipophilicity.

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4 Synthesis of Novel Nitrogen Heterocycles via Multicomponent Reactions

4.1 A Microwave-Assisted Multicomponent Synthesis of Substituted 3,4-Dihydroquinazolinones (Paper V)

4.1.1 Background and Aim In this thesis, MCRs have been used to prepare a wide range of compounds, starting from sulfonyl azides or aryl iodides. Given their atom-efficiency and operational simplicity, MCRs are of great value in the radiochemistry setting, but their chief area of use is in the synthesis of biologically active heterocyclic molecules. Although the number of synthetically tractable molecules in the “chemical universe” is vast,†††† therapeutically useful compounds appear to cluster in “galaxies”,203 slightly lessening the burden of the medicinal chemist seeking to explore new regions. Notwithstanding this, the task of probing these regions is still formidable, and new tools are required that provide rapid access to structurally diverse molecules. Therefore, a multicomponent reac-tion manifold that provides rapid access to structurally diverse molecules would be of great value, and given the biological activity attributed to the 3,4-dihydroquinazolinone skeleton, this scaffold was chosen as the synthetic tar-get.

In this Section, a common intermediate has been subjected to aza-Henry/Mannich-like conditions, leading to novel 3,4-dihydroquinazolinones with 5 points of diversity (see Scheme 12).

†††† If a list of just 150 substituents and mono- to 14-substituted hexanes is used, there are more than 1029 derivatives of n-hexane alone.

69

Scheme 12. Elaboration of cyclic iminium ion with representative 3,4-dihydro-quinazolinones underneath. Adapted from Paper V.

4.1.2 Initial Discovery and Optimisation of Reaction Parameters During the development of a Henry reaction protocol with the purpose of forming novel nitroalkenes, a hitherto unknown 3,4-dihydroquinazolinone was formed (compound 17a, see Scheme 13). This discovery provided a con-venient entry point into a useful class of heterocycles with potential for down-stream functionalisation. As previously mentioned, the nitro moiety is a syn-thetically useful intermediate, allowing access to nitrogen moieties in varying oxidation states. Furthermore, the C-2 carbonyl moiety can also undergo deri-vatisation, providing a potential site for substitution via cross-coupling reac-tions.

Scheme 13. Serendipitous discovery of one-step synthesis of 3,4-dihydroquinazoli-nones.

Due to the known thermal instability of tert-butyl carbamates204 and potential steric effects from the tert-butyl group, methyl carbamate 16a‡‡‡‡ was selected as the starting point in the microwave-assisted multicomponent synthesis of 3,4-dihydroquinazolinones (see Table 11).

‡‡‡‡ Synthesised in two steps from the corresponding aminobenzyl alcohol (see Paper V).

70

Table 11. Investigation of reaction conditions for the cyclisation of 16a using MeNO2 and NH4OAc

Entry solvent Yielda (%)

1 methanol 71 2 ethanol 79 3 acetic acid 81 4 acetic acid 71b(88)c 5 water 48 6 THF -d

aIsolated yields, 10 equiv. MeNO2 used unless otherwise stated. b2 equiv. nitrome-thane. c1 mmol scale. dNo product detected by LC/MS.

Gratifyingly, an isolated yield of 71% was achieved using methanol, which increased to 79% with ethanol, possibly due to increased solubility of 16a (Table 11, entries 1 and 2). Switching to acetic acid gave an excellent yield of 81% (entry 3), and reducing the amount of nitromethane greatly simplified purification, furnishing 17a in 71% isolated yield (entry 4). A fair yield of 48% was achieved using water as the solvent (entry 5), but using THF, no product could be detected by LC/MS.

4.1.3 Reaction Scope and Further Synthetic Applications With the optimised conditions in hand, a single reactant replacement (SRR) approach was then adopted to gauge the effect of varying nucleophiles and substituents on the o-formyl carbamate component, introducing five points of diversity to dihydroquinazolinones 17b–17l (see Table 12 overleaf).

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Table 12. Amine and aryl substrate scopea

aIsolated yields. b60 min heating. c4 equiv. MeNO2 used. dOne-pot, two-step proce-dure. eDiastereomeric mixture (dr 1.25:1, determined by 1H NMR). f4 equiv. ethyl cyanoacetate used.

Gratifyingly, anilines were also tolerated despite their reduced nucleophilicty, and 3-aminophenyldihydroquinazolinone 17c was isolated in a fair yield of 40% (17d). Next, the effect of varying the aryl substituent on the o-formyl carbamate compound (16b–16d) was investigated. Although introduction of a trifluoromethyl group required prolonged reaction times and a slight excess of nitromethane, a fair yield of 62% was obtained for 17e. Heterocyclic o-formyl carbamates could also be used, with 17f being isolated in a good yield of 61%. Electron-donating substituents on the arene performed excellently and o-formyl carbamate 16d was smoothly transformed into dihydroquinazoli-none 17g in an excellent yield of 91%.

Next, the effect of varying the carbon nucleophile was investigated, with NH4OAc as the nitrogen source. Compound 17h was isolated in a good yield

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of 72%, demonstrating a tolerance for steric effects in the carbon nucleophile. Diethyl malonate also performed well, albeit with a slight alteration to the experimental procedure whereby NH4OAc was first heated with 16a, after which the carbon nucleophile was added to give 17i in 76% yield after heating. Single crystal X-ray analysis of 17i provided final confirmation of the 3,4-dihydroquinazolinone structure (see Table 12). The one-pot, two-step proce-dure was also applied using ethyl cyanoacetate, providing access to masked β-amino acid 17j in an excellent yield of 90%.

Finally, the effect of N-1 substitution on the o-formyl carbamate was ex-amined. Gratifyingly, the introduction of a butyl substituent was not detri-mental, and 17k was isolated in 80% yield. A slightly reduced yield of 72% was obtained for the corresponding allyl compound, but overall, results were comparable to those of the parent o-formyl carbamate (16a).

As previously mentioned, the nitro moiety can act as a masked amine, and to demonstrate the utility of this synthetic protocol, compound 17a was sub-jected to further derivatisation (see Scheme 14). Catalytic hydrogenation of 17a gave amine 18 in quantitative yield. After acylation with chloroacetyl chloride, base-mediated cyclisation gave praziquantel174 analogue 20. Finally, the N-Boc protected amine 21 was prepared from 18. This synthetic sequence demonstrates the ease with which complex 3,4-dihydroquinazolinones can be synthesised from simple starting materials, and thereafter subjected to down-stream functionalisation in the search for biologically interesting molecules.

Scheme 14. Downstream functionalisation of 17a, leading to praziquantel analogue 20. Adapted from Paper V. Reagents and conditions: (a) H2, Pd/C, MeOH, r.t., 5 h; (b) pyridine, chloroacetyl chloride, 0 °C to r.t., 18 h; (c) Cs2CO3, NaI, DMF, 65 °C, 2 h; (d) Et3N, Boc2O, 0 °C to r.t., 24 h.

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4.1.4 Exploring the Reaction Pathway Based on the results above and the X-ray crystal structure obtained for com-pound 17i, the reaction is believed to proceed by the pathway shown in Figure 19. The reaction begins with formation of imine b from the amine nucleophile and o-formyl carbamate 16a. Intramolecular attack of the imine nitrogen on the carbonyl carbon of the pendant carbamate, followed by loss of methanol generates the cyclic iminium ion c. Finally, attack on the electrophilic carbon in c by an enol nucleophile generates final product d. This was supported by ESI-MS and NMR evidence showing the formation of cyclic aldimine c, and its subsequent transformation into d (evidenced by the disappearance of the characteristic imine proton of c).

Figure 19. Proposed reaction pathway for formation of 3,4-dihydroquinazolinones

4.1.5 Summary and Future Outlook (Paper V) In summary a novel microwave-assisted metal-free synthesis of 3,4-dihydro-quinazolinones has been reported. A wide range of primary amine and carbon nucleophiles were tolerated, and heterocyclic, electron-poor and electron-rich o-formyl carbamates were successfully transformed into the corresponding 3,4-dihydroquinazolinones in yields of up to 95%. The developed methodol-ogy was then applied in the synthesis of a praziquantel analogue, showcasing its versatility and utility in real-world synthetic challenges.

The discovery of a highly reactive iminium ion intermediate in Paper V paved the way for further synthetic efforts, demonstrated in Papers IX and XI. In Paper IX, unactivated ketones were utilised as nucleophiles, providing rapid access to polyfunctionalised 3,4-dihydroquinazolinones. These then un-derwent further derivatisation to yield novel polycyclic scaffolds. In Paper XI, Starting from a key iminium precursor, 13 different skeletal frameworks, including 7-membered rings and spirocyclic compounds, were accessed in a single step via adjustment of the nucleophile(s) and reaction conditions. Downstream functionalization of these molecules was also demonstrated, proving the utility of this method in accessing structurally diverse chemical entities. Furthermore, biological testing revealed a new class of anticancer compounds, easily accessed from simple starting materials. Taken together, these findings demonstrate the synthetic utility of the highly reactive iminium

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intermediate first described in Paper V. Future work in the field should be directed towards the development of asymmetric protocols, which will pro-vide unprecedented access to high-value drug compounds, including non-nu-cleoside reverse-transcriptase inhibitors of HIV.205

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5 Concluding Remarks

The overall aim of the work in this thesis was to develop methodology for the 11C-labelling of angiotensin II receptor subtype 2 (AT2R) agonists via multi-component synthesis. The specific results and conclusions are as follows:

In Paper I, an imidazole-1-sulfonyl azide salt was used to trans-form sulfonamides into sulfonyl azides. A broad range of (het-ero)aryl sulfonamides was converted into the corresponding sul-fonyl azides, including three registered pharmaceuticals, showcas-ing the potential of this methodology in late-stage azide introduc-tion. Mechanistic studies using 15N-labelled sulfonyl azides confirmed that the reaction proceeds via a diazotransfer mecha-nism.

Having demonstrated a novel method of synthesising sulfonyl az-ides, these were subjected to optimised carbonylative conditions developed in Paper II. Using PdCl2 and in situ-generated CO, a sulfonyl isocyanate intermediate was successfully trapped using amine or alcohol nucleophiles, to generate sulfonyl ureas and sul-fonyl carbamates, respectively. Additionally, a novel sulfonamide synthesis was discovered, whereby substituted sulfonamides were formed directly from the corresponding sulfonyl azide by direct displacement of the azide anion. This was confirmed by 15N NMR labelling experiments.

With methodology in hand for the carbonylative preparation of sul-fonyl carbamates, a carbon-11 labelling protocol was developed in Paper III, building on insights gained from using isotopically un-modified CO in Paper II. This novel protocol was used for prepa-ration of 12 11C-labelled sulfonyl carbamates, and then success-fully deployed in the first 11C-labelling of Compound 21, a sulfonyl carbamate compound currently in pre-clinical trials. In vitro eval-uation of carbon-11 labelled Compound 21 showed specific bind-ing to prostate cancer cells, but small-animal PET and biodistribu-tion studies showed rapid and irreversible accumulation in the liver. However, given the potential utility of an AT2R-selective im-aging agent in individualised management of prostate cancer ther-apy, as well as imaging of other disease states, these results provide an entry point into the development of future tracers to meet this need.

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Further efforts to provide information on AT2R distribution and density using 11C-labelled radiopharmaceuticals are described in Paper IV. Three potent AT2R agonists were labelled with carbon-11 in an aminocarbonylation protocol, and of these, one was se-lected to undergo in vitro and in/ex vivo evaluation. Autoradiog-raphy showed specific binding in rat pancreas, brain and kidney cortex, but small-animal PET showed rapid accumulation in the excretory organs of a female rat. This was confirmed by biodistri-bution studies. However, this represented the first synthesis and evaluation of 11C-labelled AT2R agonists and further confirmed the utility of [11C]CO as a labelling synthon.

In Paper V, a novel multicomponent reaction protocol was devel-oped, providing rapid access to structurally diverse 3,4-dihydro-quinazolinones. Starting from o-formyl carbamates and primary amines, a highly versatile iminium ion intermediate was formed, which could then be trapped by a suitable carbon nucleophile. This methodology provides an entry point to structurally diverse and bi-ologically interesting heterocyclic compounds and demonstrates the utility of multicomponent reaction protocols as a means of building molecular diversity.

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7 Acknowledgments

This thesis is based on work carried out between 2010 and 2016 at the Division of organic pharmaceutical chemistry, Uppsala University, and Uppsala PET Centre at Uppsala University Hospital. The work described herein is in many ways a collaborative effort and I am indebted to my hard-working colleagues, without whose contributions many of the experiments would never have been carried out.

Firstly, I would like to thank my supervisor Dr. Luke Odell for taking me on as a PhD student, and for indulging me when I decided to start yet another side project with uncertain prospects of success! Your scientific curiosity and im-pressive knowledge of all things organic will be a continuing source of inspi-ration.

My assistant supervisors Prof. Mats Larhed, Prof. Gunnar Antoni, Dr. Sergio Estrada and Dr. Ola Åberg for support, encouragement and insightful com-ments. Not many doctoral students have the luxury of 5 supervisors! Also, a special thank you to Dr. Jonas “Xe” Eriksson for stimulating conversations and patient help with the Xe system. Gunilla Eriksson for helping out with all things administrative, including booking the venue for my thesis defence, which I had completely forgotten. Birgitta Hellsing for all your help with students and for making sure I got my salary and actually went on holiday. Sandra Bratt and Linda Strandenhed for helping me track down various important pieces of paper. “My” post-docs, Dr. Rajiv Sawant and Dr. Shiao Chow for being a huge source of inspiration. I am very fortunate to have been allowed to work with you and I hope I will have the opportunity to pay it forwards.

The PET crowd: I know you associate me with a certain crackling sound! The PPP people: Veronika Asplund for assisting me with the rodents and Dr. Ram Kumar Selvaraju for patiently trying to explain the inner workings of a PET camera. Prof. Anna Orlova and Bogdan Mitran for assistance with prostate tumour studies. The hard-working Uruguayans at CUDIM, thanks for expert assistance.

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New and old colleagues at OFK, fellow PhD students toiling by the sweat of their brows, the post docs and all the “new” people. Prof. Bobo Skillinghaug (FRS, VC) for careful proof-reading of this manuscript. International collaborators: Prof. John Joule of Manchester, Prof. Stanley Mukanganyama of the University of Zimbabwe, Prof. Eduardo Savio and Dr. Williams Porcal of the University of the Republic of Uruguay for hosting me during my research visit. T3P and Paper IV co-authors, thanks a heap! Dr. Jonas Sävmarker and Dr. Jonas Lindh for taking me on as a summer stu-dent. Dr. Johan Gising, for getting things done. Summer and diploma students: Gabriella Andersson, Lewend Turanli, Luay Katan and Abdullah Al-Ramahi. Dr. Lisa Haigh of the Imperial College Mass Spectrometry Service for MS magic and Dr. Anatoly Mishnevs of the Latvian Institute of Organic Synthesis for your crystallography juju. Also Dr. Ewa Rozycka-Sokolowska, pity it was the wrong regioisomer! Prof. Adolf Gogoll for assistance with 15N NMR. My HUGE Swedish and Spanish and African families, there’re so many of you. My three horrible siblings (Bren, keep the hippie flame burning, and twins, stop growing!) and my fearsome mother. The Hornudden crowd- al-ways working! >1000 failed experiments, 3 children, a few close calls and a rat bite later, I think, my dear wife Viveca Sjöstedt, it’s time for me to find a safer hobby.

Marc Yendall Stevens

In the shadow of Mt. Sainte-Victoire, August 2016

EX ARDUIS FLORIO

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A doctoral dissertation from the Faculty of Pharmacy, UppsalaUniversity, is usually a summary of a number of papers. A fewcopies of the complete dissertation are kept at major Swedishresearch libraries, while the summary alone is distributedinternationally through the series Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty ofPharmacy. (Prior to January, 2005, the series was publishedunder the title “Comprehensive Summaries of UppsalaDissertations from the Faculty of Pharmacy”.)

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