Synthesis of Substituted Ring- Fused 2-Pyridones and Applications in Chemical Biology

Home , Kurt Alder

Christoffer Bengtsson

Doctoral thesis

Department of Chemistry Umeå University Umeå, Sweden 2013

Christoffer Bengtsson

Title

Synthesis of Substituted Ring-Fused 2-Pyridones and Applications in Chemical Biology

Abstract

Antibiotics have been extensively used to treat bacterial infections since Alexander Fleming’s discovery of penicillin 1928. Disease causing microbes that have become resistant to antibiotic drug therapy are an increasing public health problem. According to the world health organization (WHO) there are about 440 000 new cases of multidrug-resistant tuberculosis emerging annually, causing at least 150 000 deaths. Consequently there is an immense need to develop new types of compounds with new modes of action for the treatment of bacterial infections. Presented herein is a class of antibacterial ring-fused 2- pyridones, which exhibit inhibitory effects against both the pili assembly system in uropathogenic Escherichia coli (UPEC), named the chaperone usher pathway, as well as polymerization of the major curli subunit protein CsgA, into a functional amyloid fibre. A pilus is an organelle that is vital for the bacteria to adhere to and infect host cells, as well as establish biofilms. Inhibition of the chaperone usher pathway disables the pili assembly machinery, and consequently renders the bacteria avirulent. The focus of this work has been to develop synthetic strategies to more efficiently alter the substitution pattern of the aforementioned ring- fused 2-pyridones. In addition, asymmetric routes to enantiomerically enriched key compounds and routes to compounds containing BODIPY and coumarin fluorophores as tools to study bacterial virulence mechanisms have been developed. Several of the new compounds have successfully been evaluated as antibacterial agents. In parallel with this research, manipulations of the core structure to create new heterocycle based central fragments for applications in medicinal chemistry have also been performed.

Keywords

Synthesis, 2-pyridone, 2-thiazoline, cross coupling, pili, curli, antibacterial

Table of Contents

Table of Contents ii List of Papers iii Abbreviations v Introduction 1 History of organic synthesis: a Nobel Prize odyssey 1 Thiazolino ring-fused 2-pyridones 3 Biological target: the chaperone usher pathway 4 Biological target: curli inhibition 7 Biological testing for pilicide/curlicide activity 8 Synthetic development of the bicyclic 2-pyridones (Paper I + II) 9 Suzuki⎯Miyaura couplings onto bicyclic 2-pyridones (Paper I) 9 Synthesis of a bromomethyl substituted scaffold (Paper II) 14 Fluorescence: lighting up bacterial virulence (Paper III) 21 Synthesis of the coumarin analogues 21 Synthesis of the BODIPY analogues 26 Triazoles (Paper IV) 31 Functionalization of the 8-position 32 Functionalization of the 2-position 34 Acetylene spacer analogues (Paper V) 37 Asymmetric synthesis of Δ2-thiazolines (Paper VI) 43 2-Furanone or 2-pyrone ring-fused tricyclic scaffolds (Paper VII) 57 Concluding remarks 66 Acknowledgements 67 References 68

ii List of Papers

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

I. Bengtsson C., Almqvist F. Regioselective Halogenations and Subsequent Suzuki-Miyaura Coupling onto Bicyclic 2-Pyridones J. Org. Chem., 2010, 75, 972-975

II. Chorell E., Bengtsson C., Sainte-Luce Banchelin T., Das P., Uvell H., Sinha A. K., Pinkner J. S., Hultgren S. J., Almqvist F. Synthesis and Application of a Bromomethyl Substituted Scaffold to be Used for Efficient Optimization of Anti-Virulence Activity Eur. J. Med. Chem., 2011, 46, 1103-1116

III. Chorell E., Pinkner J. S., Bengtsson C., Edvinsson S., Cusumano C. K., Rosenbaum E., Johansson L. B. Å., Hultgren S. J., Almqvist F. Design and Synthesis of Fluorescent Pilicides and Curlicides: Bioactive Tools to Study Bacterial Virulence Mechanisms Chem. Eur. J. 2012, 18, 4522-4532

IV. Bengtsson C., Lindgren A. E. G., Uvell H., Almqvist F. Design, Synthesis and Evaluation of Triazole Functionalized Ring-Fused 2- Pyridones as Antibacterial Agents Eur. J. Med. Chem., 2012, 54, 637-646

V. Andersson E. K., Bengtsson C., Evans, M. L., Chorell E., Sellstedt M., Lindgren A. E. G., Hufnagel D. A., Bhattacharya M., Tessier P., Wittung-Stafshede P., Almqvist F., Chapman M. R. Modulation of Curli Assembly and Pellicle Biofilm by Chemical and Protein Chaperones 2013, Manuscript

VI. Bengtsson C., Nelander H., Almqvist F. Asymmetric Synthesis of 2, 4, 5-Trisubstituted ∆2-Thiazolines Chem. Eur. J., 2013, DOI: 10.1002/chem.201301120

VII. Bengtsson C., Almqvist F. A Selective Intramolecular 5-exo-dig or 6-endo-dig Cyclization en Route to 2-Furanone or 2-Pyrone Containing Tricyclic Scaffolds J. Org. Chem., 2011, 76, 9817-9825

Reprints have been made with permission from the respective publisher.

iii Other papers by the author not appended to this thesis

Horvath I., Weise C. F., Andersson E. K., Chorell E., Sellstedt M., Bengtsson C., Olofsson A., Hultgren S. J., Chapman M. R., Wolf-Watz M., Almqvist F., Wittung-Stafshede P. E. L. Mechanisms of Protein Oligomerization: Inhibitor of Functional Amyloids Templates α-Synuclein Fibrillation J. Am. Chem. Soc., 2012, 134, 3439-3444

Chorell E., Pinkner J. S., Bengtsson C., Sainte-Luce Banchelin T., Edvinsson S., Linusson A., Hultgren S. J., Almqvist F. Mapping Pilicide Anti-Virulence Effect in Escherichia coli, a Comprehensive Structure-Activity Study Bioorg. Med. Chem., 2012, 20, 3128-3142

iv Abbreviations

Aβ Amyloid β

Ac Acetyl

AD Asymmetric dihydroxylation aq Aqueous

Arg Arginine

Bn Benzyl

Boc tert-butyloxycarbonyl

BODIPY 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene

Bu Butyl cat. Catalytic conc. Concentrated cPr Cyclopropyl

CuAAC Copper(I) catalyzed azide alkyne cycloaddition

CuTC Copper(I) thiophene-2-carboxylate

DCC N,N´-dicyclohexyl carbodiimide

DCE 1,2-dichloroethane

DCM Dichloromethane

DMAP 4-dimethylaminopyridine

DMF N,N-dimethylformamide

DMPK Drug metabolism and pharmacokinetics

v DMPU 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone

DMSO dimethylsulfoxide

DPPF 1,1’-bis(diphenylphosphino)ferrocene

DPPP 1,3-bis(diphenylphosphino)propane

E. coli Escherichia coli e.g. Exempli gratia (Latin for ”for example”)

EC50 Half maximal effective concentration ee Enantiomeric excess eq Equivalent

Et Ethyl et al. et alia (Latin for ”with others”)

FGI Functional group interconversion h hour

N,N,N´,N´-tetramethyl-O-(1H-benzotriazol-1- HBTU yl)uranium hexafluorophosphate

HMDS 1,1,1,3,3,3-hexamethyldisilazane i.e. id est (Latin for ”that is”) in vitro Latin for ”in glass”

LDA Lithiumdiisopropylamide

LiHMDS Lithiumhexamethyldisilazane

LR Lawesson’s reagent

Lys Lysine

vi mCPBA meta-chloroperoxybenzoic acid

Me Methyl

MeCN Acetonitrile

Meldrum’s acid 2,2-dimethyl-1,3-dioxane-4,6-dione

MeOH Methanol

MS Molecular sieves

Ms Methanesulfonyl

MWI Microwave irradiation

NHC N-heterocyclic carbene

NIS N-iodosuccinimide

NMO N-Methyl morpholine N-oxide

NMR Nuclear magnetic resonance

Ns para-nitrobenzenesulfonyl

Pd/C Palladium on charcoal

Pd-NHC or Pd-IPr- [1,3-Bis(2,6-Diisopropylphenyl)imidazole-2- NHC ylidene](3-chloropyridyl)palladium(II) dichloride

[1,3-Bis(2,6-diisopropylphenyl)imidazolidene](3- Pd-SIPr-NHC chloropyridyl) palladium(II) dichloride

Ph Phenyl

5-Phenyl-2-[4-(5-phenyl-1,3-oxazol-2-yl)phenyl]-1,3- POPOP oxazole quant quantitative ref Reference

vii rt Room temperature

(s) Solid state

TBAF Tetrabutylammonium fluoride

N,N,N´,N´-tetramethyl-O-(1H-benzotriazol-1- TBTU yl)uronium tetrafluoroborate

TEA Triethylamine

Tf Trifluoromethanesulfonyl

TFA Trifluoroacetic acid

THF Tetrahydrofuran

ThT Thioflavin T

TMS Trimethylsilyl

TMSA Trimethylsilylacetylene

TMSE Trimethylsilylethyl

UPEC Uropathogenic Escherichia coli

WHO World health organization

Å Ångström

≠ is not equal to

viii

Introduction

History of organic synthesis: a Nobel Prize odyssey The word synthesis originates from the ancient Greek word σύνθεσις, which means “composition” that in this case, refers to the joining of one or more entities together to create something new. Friedrich Wöhler´s synthesis of urea in 1828,1 followed by Hermann Kolbe´s synthesis of acetic acid in 18492 can be considered as the beginning of organic synthesis (Figure 1). At the end of the 19th century, Adolf von Baeyer contributed to organic chemistry by his synthesis of the blue colored dye indigo (Figure 1). Adolf von Baeyer also discovered the oxidation procedure known as the Baeyer-Villiger oxidation, together with the Swiss chemist Victor Villiger at the end of the 19th century.3 In 1905, Adolf von Baeyer received the Nobel Prize in chemistry, partly for his work with indigo. During the 20th century, incredible development has occurred in organic synthesis. In 1900 the French chemist Victor Grignard made his breakthrough discovery of how to make carbon-carbon bonds from an organic halide, magnesium metal and a ketone,4 a very important discovery in organic synthesis indeed. Together with Paul Sabatier, he earned the Nobel Prize in chemistry in 1912 for his achievements in hydrogenations of organic compounds in the presence of finely disintergrated metals. In 1928 Otto P. H. Diels and Kurt Alder discovered their novel reaction for the construction of six membered rings, known as the Diels-Alder reaction.5 The Diels-Alder reaction is today widely used in organic synthesis to construct very complex cyclic systems from simple starting materials.6 Otto P. H. Diels and Kurt Alder received the Nobel Prize for chemistry in 1950 “for their discovery and development of the diene synthesis”. In 1954 Robert B. Woodward was the first to synthesize the alkaloid strychnine7 (Figure 1) and earned the Nobel Prize for chemistry in 1965 for his outstanding achievements in organic synthesis. In the fifties and sixties George Wittig and Herbert C. Brown contributed to the development of synthetic organic chemistry with their respective work on phosphorus8 and boron9 containing compounds. They were jointly awarded the Nobel Prize for chemistry in 1979 for their achievements. George A. Olah also began his work on carbocations and their use in organic synthesis in the fifties10 in work that earned him the Nobel Prize for chemistry in 1994. In the early sixties R. Bruce Merrifield11 founded his polymer supported technique for peptide synthesis. The techniques he pioneered are still used today in peptide synthesis as well as in polymer supported reagents and scavengers. R. Bruce Merrifield was awarded the Nobel Prize in chemistry in 1984 “for his development of methodology for chemical synthesis on a solid matrix”. During the seventies Jay K. Kochi (1971),12 Tsutomu Mizoroki (1971),13 Richard F. Heck (1972),14 Robert J. P. Currio (1972),15 Makoto Kumada

(1972),16 Kenkichi Sonogashira (1975),17 Ei-ichi Negishi (1977)18, John K. Stille (1978),19 Norio Miyaura (1979)20 and Akira Suzuki (1979)20 revolutionized synthetic organic chemistry with their transition metal catalyzed reactions. Richard F. Heck, Ei-Ichi Negishi and Akira Suzuki were awarded the Nobel Prize in chemistry, 2010, for their amazing work with palladium catalyzed cross couplings in organic synthesis. Their respective methods are today well-established tools among synthetic organic chemists and are widely applied on a routine basis.21 The areas of asymmetric synthesis and metathesis were established during the end of sixties and through to the beginning of the eighties. William S. Knowles,22 Ryoji Noyori23 and K. Barry Sharpless24 earned the Nobel Prize for chemistry in 2001 for their outstanding work with asymmetric hydrogenations (Knowles and Noyori) and asymmetric oxidations (Sharpless) respectively. Their methods are today frequently used in organic synthesis laboratories around the world.25 In 2005 Yves Chauvin,26 Robert H. Grubbs27 and Richard R. Schrock28 were awarded the Nobel Prize in chemistry for their work developing metathesis reactions in organic synthesis, which began in the seventies. This technique for coupling two alkenes with each other under transition metal catalysis is today widely used in research laboratories29 as well as in industry.30 In 1990, Elias J. Corey was awarded the Nobel Prize in chemistry “for his development of the theory and methodology in organic synthesis”. He has contributed extensively to the development of many reactions (Corey⎯Bakshi⎯Shibata reduction, Corey⎯Chaykovsky reaction, Corey⎯Fuchs reaction, Corey⎯House reaction, Corey⎯Kim oxidation, Corey lactonization, Corey⎯Nicolaou macrolactonization, Corey⎯Seebach reaction, Corey⎯Winter olefination) as well as to the area of retrosynthetic analysis. Finally, in an example of the complexity synthetic organic chemists can create in the laboratories today, in 1995 after twelve years of work, K. C. Nicolaou et al. completed the total synthesis of the extremely complex marine toxin Brevetoxin B31 (Figure 1) with Matsuo et al. publishing a refined version of this synthesis in 2004.32 Despite all the Nobel Prizes and all the tremendous developments in organic synthesis throughout the years, considering the infinite number of possible organic molecules, there is still a great demand for new developments and refinements to synthetic methodologies, which will be revealed in this thesis.

N O O O H N H H N NH 2 2 OH N N H Urea Acetic acid H Friedrich Wöhler O O Hermann Kolbe H O Indigo Adolf von Baeyer Strychnine Robert B. Woodward

HO O

H H O O H H H H O O H H H O O O H O O H H O O O H H H H Brevetoxin B K. C. Nicolaou Figure 1. The development in the complexity of synthetic organic molecules since the 1800s.

Thiazolino ring-fused 2-pyridones The 2-pyridone core is an interesting heterocycle which is common to many natural products (Figure 2) as well as synthetic compounds, which possesses widespread biological characteristics. The 2-pyridone core is also present in ® ® registered pharmaceuticals: for instance Corotrop and Primacor , that are both used in the treatment of heart failure. The active ingredient in these drugs is the 2-pyridone based compound milrinone (Figure 2), which is a phosphodiesterase inhibitor. Sebiprox®, used in treatment of dandruff, also contains a 2-pyridone based active ingredient called ciclopirox (Figure 2).

HO O OH O N N O N O H Camptothecin OH O Pretenellin B

O N N N NH N O N O H OH Cytisine Milrinone Ciclopirox

Figure 2. Examples of naturally occurring 2-pyridones, together with two examples of 2-pyridone based compounds incorporated in registered pharmaceuticals

A well-known application area for 2-pyridones is as peptidomimetics,33 and thiazolino ring-fused 2-pyridones were initially designed for this purpose.33e When the project presented in this thesis began in 2008, the synthetic pathway to the thiazolino ring-fused 2-pyridones was well established (Scheme 1)33e, 34 and many compounds with variations mainly in the 3-35, 6- ,33i, 36 and 8-positions34b had been synthesized.

R HO

O O O O a O O O O R1 1 7 8 d R S 2 1 6 N 3 R 5 4 S O O Cl NH O 1 b 2 c R 1 N N R EtO O O

Scheme 1. Established synthetic route for thiazolino ring-fused 2-pyridones. Conditions: a) RCH2COOH, DCC, DMAP, DCM; b) HCl (g), EtOH; c) L-Cys-Me- ester*HCl, TEA, DCM; d) TFA, MWI 140 oC 2 min, DCE.

Some compounds with substituents in position 2 of the thiazoline ring had been synthesized, however only as racemic mixtures.37 Not much alteration to either the central fragment38 or the naphthyl substituent in position 734b had been performed. Furthermore it was known that the biological target of these peptidomimetic ring-fused 2-pyridones in E. coli could be altered from inhibiting pili assembly to preventing curli assembly by altering the substitution patterns, particularly in the 8-position.39 Consideration had also been given to making compounds with fluorescent properties by adding tags such as BODIPY or coumarin to the central fragment to study the bacterial virulence mechanisms further, and modifying the central fragment to access novel heterocyclic ring-fused central fragments.

Biological target: the chaperone usher pathway Considering the ever-growing bacterial resistance there is an immense need to develop new methods to treat bacterial infections.40 Traditional methods of antibiotic discovery have failed to keep pace with the evolution of this resistance. The pilus is an organelle that is vital for the bacteria in order to adhere to and infect host cells, as well as in establishing biofilms. Disabling these organelles renders the bacteria avirulent. This will at least in theory lead to more tolerated antibiotics, and hence slower development of resistance. It has been known for several years that the ring-fused 2- pyridones exhibit biological activity against the pili assembley machinery in

uropathogenic E. coli, termed the chaperone usher pathway.33e, 39b The chaperone usher pathway is a complex cascade reaction which occurs in the periplasm of Gram-negative bacterium. There are many different types of pili, of which the Type 1 and P-pili involved in the infection of bladder cells and kidneys are the most studied.41 Pili are built up from repeating protein subunits; these subunits are called Pap in P-pili and Fim in Type 1 pili. One of the key proteins in the assembly of P pili is the chaperone PapD, which is responsible for the delivery of the pili building blocks from the surface of the inner membrane to the assembly area located at the outer membrane (Figure 3).

Figure 3. Schematic view over the chaperone usher pathway (illustration by Scott Hultgren lab, Washington University, St. Louis, Missouri, USA)

An X-ray structure of PapD together with a C-terminal peptide part (Figure 4) from the pilus adhesin PapG was the inspiration to develop bicyclic 2- pyridone based peptidomimetics.33e The structure revealed that the C- teminus of the peptide formed key interactions with the Arg 8 and Lys 112 residues of the chaperone PapD active site.42

SMe

O O O H H H N N N N 2 N N N H H H O O O COOH OH OH

S N O COOH Figure 4. The C-terminal peptide shown to bind to the active site of PapD: the source of inspiration for thiazolino ring-fused 2-pyridone based peptidomimetics.

Several peptide analogues and thiazolino ring-fused 2-pyridones were synthesized and their binding ability to PapD measured by surface plasmon resonance. These type of studies do not provide information on where the ligand is binding on the protein, and thus a complementary dissociation study was performed with the FimC-FimH complex. The 2-pyridone shown in Figure 4 was the most promising and was able to almost completely dissociate the FimC-FimH complex at a 150-fold excess.33e This pilicide was subsequently evaluated for its ability to inhibit pili growth by comparing treated and non treated bacteria under atomic force microscopy (Figure 5).

Figure 5. Atomic force microscopy images of A) untreated bacteria B) bacteria treated with the illustrated 2-pyridone compound. Complete depiliation at 3.6 mM (pictures by Dr J Jass).

Significantly, despite the high compound concentration used in this study, bacterial growth was unaffected. Today we have ring-fused 2-pyridones with 43 EC50 ≤ 400 nM.

Biological target: curli inhibition This project originated from earlier reports on tricyclic compounds containing the 2-pyridone core (e.g. structure A, Figure 6) which were reported to have effect against Aβ-peptide aggregation implicated in Alzheimer’s disease.44 As these compounds did not contain a carboxylic acid substituent, synthetic pathways to the decarboxylated versions of the thiazolino ring-fused 2-pyridones were developed (structure B, Figure 6).45 These compounds were subsequently investigated for their ability to inhibit Aβ-peptide aggregation. For thiazolino ring-fused 2-pyridones, the carboxylic acid substituent in the C-3 position was found to be mandatory for inhibitory activity, with neither the decarboxylated compounds or the esters active.45

N H N O S R1

O S N N

O O A B

Figure 6. A) Example of a reported tricyclic 2-pyridone with inhibitory effects on Aβ-peptide aggregation. B) General structure of the prepared decarboxylated ring- fused 2-pyridone analogues.

Amyloid fibres are normally β-sheet rich structures and ring-fused 2- pyridones were originally designed as C-terminal β-sheet mimetics.33e Considering this, our previous success with pili inhibition, and the reported inhibitory effects of 2-pyridones (structure A, Figure 6) on Aβ-peptide aggregation,44 we directed our attention towards the curli fibres. Curli are functional extracellular amyloid fibres produced by uropathogenic E. Coli. These extracellular fimbriae enable biofilm formation and promote pathogenicity. Two examples of thiazolino ring-fused 2-pyridones were investigated for their ability to prevent curli fibre formation. This by preventing polymerization of the major curli subunit protein CsgA.39a Furthermore it was found that the substituent, especially in the 8-position of the ring-fused 2-pyridone scaffold was important for selectivity for inhibition

of CsgA polymerization. Like the pilicides, the curlicides have also been evaluated under electron microscope to characterise the biological effect (Figure 7).

Figure 7. Electron microscope images of A) untreated bacteria B) bacteria treated with the depicted compound. Complete decurliation was observed at a compound concentration of 125 µM (pictures by Scott hultgren lab, Washington University, St. Louis, Missouri, USA).39a

Biological testing for pilicide/curlicide activity

Biological testing for pili and curli biofilm formation presented in this thesis were performed in the respective assays described below. For all analyses, the clinical E. Coli isolate UTI89 was used and all measurements were performed in triplicates. The growth conditions utilized in the different assays are known to preferably express pili and curli respectively. The ability to block pili dependent biofilm formation was measured in polyvinylchloride 96-well plates. Bacteria were grown with and without compound present, and the biofilm formed on the bottom of the well coloured by crystal violet. After washing and drying the crystal violet bound to the biofilm was dissolved by acetic acid and absorbance measured at 600 nm. The absorbance of the crystal violet is proportional to the amount of biofilm formed. In parallel to this a bacterial growth study was performed to determine compound toxicity. This was measured by absorbance spectroscopy at 600 nm on cells grown in the presence of compound in polystyrene wells. The ability to inhibit curli dependent biofilm formation was measured in an assay similar to the pili assay described. The biofilm was grown under curli dependent conditions, with the biofilm formed in the liquid/air interface instead of the bottom of the well. This assay is normally refered to as the pellicle assay.

Synthetic development of the bicyclic 2- pyridones (Paper I + II)

The typical thiazolino ring-fused 2-pyridone synthetic sequence consists of between 5 to 7 steps depending on whether you need to synthesize the nitrile or not (Scheme 1). This synthetic sequence is robust, typically high yielding and amenable to scaling up. However it suffers from the fact that the R and R1 substituents are introduced at the beginning of the synthesis, which makes divergent library synthesis very time consuming. The aim of these two papers was to develop more efficient ways to alter the substitution pattern of the ring-fused 2-pyridones in positions 8 and 7 respectively.

Suzuki⎯Miyaura couplings onto bicyclic 2-pyridones (Paper I)

The substituent in the 8-position of the bicyclic 2-pyridones has been shown to be important in achieving selectivity between pili and curli inhibitory effects.39 In order to investigate this further, it was desirable to develop a scaffold that could be substituted later in the synthetic sequence. This preferably could be achieved via the Suzuki⎯Miyaura coupling onto a corresponding halide. The Suzuki⎯Miyaura coupling is robust, uses non- toxic boronic acids/esters, tolerates a variety of functional groups and solvent systems and is scalable,46 and today is used extensively in the synthesis of drugs46-47 and natural products.48 Due to the popularity of the Suzuki⎯Miyaura coupling, a large variety of boronic acids/esters are commercially available. These features make the Suzuki⎯Miyaura coupling ideal for library syntheses. The Suzuki⎯Miyaura coupling is traditionally a reaction between a vinylic halide/triflate and an sp2 hybridized boronic acid/ester under palladium catalysis.49 In recent years Gregory C. Fu and coworkers has developed this reaction to make it possible to also couple two sp3 hybridized carbons in good yields.50 After consulting the literature we envisioned a synthetic pathway to 2 starting from the already known ring- fused 2-pyridone 133e (Scheme 2, part A). Initially, a selective iso- amylnitrite/HBr promoted halogenation procedure onto 1 was performed, which had been previously used for similar compounds (Scheme 2, part B).51

Br A S a S S N N N Br O O O O O O O O O 1 2 3

O O O O Br B b N N

O O O O O O

Scheme 2. Halogenation of ring-fused 2-pyridones. Conditions: a) 2 eq iso- amylnitrite, 1 eq conc. HBr (aq), DCM, -40 to 15 oC, 6 h, 85%; b) 2 eq iso-amylnitrite, 1 eq conc. HBr (aq), DCM, 20 oC, 4 h, 78%

With our more electron rich system this reaction gave compound 3 in 85% yield with complete regioselectivity.52 Initially it was not obvious that compound 3 was formed and extensive 1D and 2D NMR studies to assign the structure were inconclusive. Instead, a Suzuki⎯Miyaura coupling with phenylboronic acid was performed on the product and the spectral data compared with literature values for the 8-substituted derivative,33e which was shown to be a mismatch. The difference in regioselectivity for the thiazolino ring-fused 2-pyridones versus the carbam analogues reported in the literature may arise from the differences in resonance stabilization of the carbocation together with the electron donating properties of the sulfur substituent, which both favour bromination in the 6-position (Scheme 3).

R S R S R S R S A N N N N "Br+" Br Br Br O O O O O O O O O O O O

"Br+" Br Br

R S R S R S B N N N O O O O O O O O O

Scheme 3. Potential resonance stabilization in the bromination of thiazolino ring- fused 2-pyridones: A) 6-position bromination, B) 8-position bromination.

The reactivities of different halides/pseudohalides generally follow the trend + N2 > I > TfO > Br >> Cl in Pd catalyzed cross couplings. In addition Conreaux et al. had earlier reported similar regioselective Suzuki⎯Miyaura couplings on monocyclic bromo-iodo systems.53 Considering this we envisioned the synthesis of the bromo-iodo substituted scaffolds 6 and 7 (Scheme 4). This route would ultimately enable further substitution in the 6- position via a second cross coupling.

R S N Br c O O O

S 6 R = 1-naphthyl R a or b R S 7 R = H N N X d S O O O O R O O N 1 R = 1-naphthyl 3a R = 1-naphthyl, X = Br 4 R = H 3b R = 1-naphthyl, X = I O O O 5a R = H, X = Br 5b R = H, X = I 3c R = 1-naphthyl 5c R = H Scheme 4. Selective halogenations of ring-fused 2-pyridones 1 and 4, conditions: a) iso-amylnitrite, conc. HBr (aq), DCM, -40 to 15 oC, 6 h, (3a = 85%, 5a = 82%); b) iso- amylnitrite, conc. HI (aq), DCM, -40 to rt, 15-18 h, (3b =73%, 5b = 75%); c) NIS,

MeCN, reflux, 3.5-5 h, (6 = 91%, 7 = 85%); d) Pd(OAc)2, PhB(OH)2, KF, MeOH, MWI 110 °C 10 min, (starting from 3b and 5b gave 3c = 86%, 5c = 83%).

The synthesis of the bromo-iodo scaffolds 6 and 7 was performed via the 6- brominated analogues 3a and 5a, followed by iodination of the 8-position with NIS in refluxing MeCN. The brominated analogues 3a and 5a were evaluated in the ligand free Pd(OAc)2 catalyzed Suzuki⎯Miyaura coupling with phenylboronic acid. Unfortunately these conditions gave only about 20% conversion of the starting material. This problem could be circumvented by exchanging the bromine substituent for an iodine, with 3b and 5b accessible by exchanging HBr for HI in the halogenation procedure. The subsequent Suzuki⎯Miyaura coupling under the same ligand free conditions (Pd(OAc)2, PhB(OH)2, and KF in MeOH) afforded the desired coupling products 3c and 5c in 86% and 83% yield respectively. The synthetic pathway to the bromo-iodo scaffolds was shown to be robust and scalable, with 6 and 7 prepared on a gram scale. Couplings of bromine substituents in the 8-position of similar thiazolino ring-fused 2-pyridones was previously found to be demanding when attempted by Seger et al.54 However, with the more reactive iodo- substituents, and following the successful coupling of 3b and 5b, we

envisioned performing a regioselective cross-coupling under ligand free conditions, with the bromo-iodo scaffolds 6 and 7 (Table 1). I R1

R S a R S N N Br Br O O O O O 8 O Table 1. Selective Suzuki⎯Miyaura couplings, conditions a) R1-B(OH)2, Pd(OAc)2 or Pd-NHC, KF, MeOH, MWI 110oC for 10 min or 120 oC for 10 min. R R1 Catalysta T (oC) Yield (%) Product b 1-Naphthyl Ph Pd(OAc)2 110 66 8a b H Ph Pd(OAc)2 110 85 8b b 1-Naphthyl 4-carboxyPh Pd(OAc)2 120 45 8c b H 4-carboxyPh Pd(OAc)2 120 67 8d 1-Naphthyl 4-methoxyPh Pd-NHC 110 62 8e H 4-methoxyPh Pd-NHC 110 83 8f 1-Naphthyl 5-indolyl Pd-NHC 110 61 8g H 5-indolyl Pd-NHC 110 83 8h aPd-NHC = [1,3-Bis(2,6-Diisopropylphenyl)imidazol-2-ylidene](3- chloropyridyl)palladium (II) dichloride bUsing Pd-NHC instead of Pd(OAc)2 did not increase the yields

The more sterically demanding CH2-naphthyl substituent generally gave about 20% lower yield than its methyl counterparts. The phenyl- and 4- carboxyphenyl boronic acids were efficient coupling partners under the ligand free conditions. In the case of 4-methoxyphenyl- and 5-indolyl boronic acids, the Pd-NHC catalyst proved more effective (Table 1). The stoichiometry of the boronic acid was important: when using more than 1 eq we observed mixtures of the mono- and diarylyated products. The 6- brominated ring-fused 2-pyridones 8 prepared were interesting intermediates for further functionalization in position 6. Compounds 8a-h were evaluated in either a transfer hydrogenation with Pd/C and ammoniumformate in refluxing MeOH or a second Suzuki⎯Miyaura coupling (Table 2). Ligand free coupling conditions in this case gave poor conversion (about 20%), however exchanging the Pd(OAc)2 for Pd-NHC afforded 10 in 72% yield.

R1 R1 b S a S R R S N N N Br O O O O O O HN O O O 10 8 9

Table 2. Dehalogenation or Suzuki⎯Miyaura coupling of 8. Conditions: a) Ammoniumformate, Pd/C, MeOH reflux 1-3 h; b) Indole-5-boronic acid, Pd-NHC, KF, MWI 120 oC for 10 min, 72%. R R1 Yield (%) Product 1-naphthyl Ph 94 9a 1-naphthyl 4-carboxyPh 94 9b 1-naphthyl 4-methoxyPh 91 9c 1-naphthyl 5-indolyl 96 9d

In summary, we have developed scalable synthetic pathways to the bromo- iodo scaffolds 6 and 7, which have been evaluated in a regioselective Suzuki⎯Miyaura cross coupling together with various arylboronic acids. The remaining bromine substituent can either undergo a second Suzuki⎯Miyaura coupling if further substitution in position 6 is desired (exemplified by compound 10, Table 2), or be removed by transfer hydrogenation with ammoniumformate and Pd/C in excellent yields (Table 2). Furthermore the bromo-iodo scaffolds developed herein can also serve as useful starting materials for other cross coupling functionalizations, and have been successfully used in the Sonogashira coupling discussed in paper IV and V.55

Synthesis of a bromomethyl substituted scaffold (Paper II)

Few changes had been made to the naphthyl substituent in position 7 of the ring-fused 2-pyridones when this project began. In this work, we aimed to develop an efficient platform for the diversification of substituents in position 7. We envisioned a bromomethyl substituent in position 7 as a good starting point for this functionalization. The alkyl bromide is a diverse functional group that can be manipulated in a variaty of ways. In this paper we focused on SN2 additions with oxygen and nitrogen nucleophiles, with an additional aim to make C-C bonds via Suzuki⎯Miyaura cross coupling. For this strategy we desired the bromomethyl substituted acyl meldrum acid derivative 11, which was synthesized from bromoacetic acid, Meldrum’s acid, DCC and DMAP (Scheme 5). The purity of 11 is pivotal for the outcome of the following ketene/imine cyclocondensation and the quality of 11 varied between batches. This spurred us to develop a bromomethyl substituted dioxin-4-one based acyl ketene source (12, Scheme 5). This type of ketene source has previously been used in the ketene/imine cyclocondensation reaction with good results.52

Br HO Br O O a O O O O O O O O O 12 11 Scheme 5. The bromomethyl substituted acyl Meldrum’s acid shown together with the desired dioxin-4-one based acyl ketene source. Conditions: a) BrCH2COOH, DCC, DMAP, DCM 0 oC to rt, 18 h, 71%.

Radical bromination of the commercially available methyl substituted dioxin-4-one 13 was reported in the literature,56 as well as the deprotonation with LDA and quenching by a bromine source.57 Unfortunately neither of these strategies worked in our hands, and gave extensive bromination in the α-position of the dioxin-4-one ring (14, Scheme 6). Inspired by a recent article regarding selective γ-brominations of β-keto esters,58 we envisioned a new synthetic pathway to 12. By treating commercially available tert-butyl acetoacetate 15 with 1 eq of bromine in CHCl3, followed by bubbling air through the solution for 2 h, the γ-brominated β-keto acid 16 was obtained in 69% yield. The air bubbling was crucial to add molecular oxygen to the reaction mixture and create the best conditions for the bromine migration from the α- to the γ-position.59

Radical Br O bromination O O

O O or O O O O LDA/"Br+" 13 12 14

O O a O O b O Br O HO O O 15 16 12 Scheme 6. The unsuccessful bromination attempts shown together with the synthesis of 12. Conditions a) Br2, air, CHCl3, 0 oC to rt, 17h, 69% b) Acetone, Ac2O, cat. conc. H2SO4, rt, 3h, quant

The γ-brominated β-keto acid 16 must be handled and purified with care, and used quickly in the next reaction to prevent decarboxylation. Despite this, there are some advantages with the dioxin-4-one based acyl ketene source in comparison to the acyl Meldrum’s acid derivatives. Firstly, they are stable enough to purify if required by column chromatography on silica gel.

Secondly, they do not liberate CO2 (g) when decomposing to the corresponding acyl ketene. Hence more concentrated reactions can be performed under microwave heating, which is beneficial for large scale synthesis. With the bromomethyl substituted acyl ketene sources 11 and 12 in hand we could synthesize the desired ring-fused 2-pyridones 18 (Scheme 7) in gram quantities.

Br 1 Br 1 R HO R S Br S O O or O a N N O O O O O O O O 12 O 1 11 17a R = cPr 18a R1 = cPr 17b R1 = mCF Ph 1 3 18b R = mCF3Ph

Scheme 7. Synthesis of the desired bromomethyl substituted ring-fused 2- pyridones. Conditions: a) TFA, DCE, MWI 140 °C for 2 min, 18a from 11 = 87%; from 12 = 64%; , 18b from 11 = 92%; from 12 = 74 %

The bromomethyl substituted compounds 18 were evaluated in SN2 substitutions with three different secondary amines (Table 3). In addition, the primary amines were synthesized via azide substitution followed by a Zn

(s)/NH4Cl mediated reduction in EtOH:H2O (3:1). Primary amines 21a and 21b were then further reacted with either acid chlorides or sulfonyl chlorides

to access the corresponding amides 22a-d and sulfonamides 23a-d (Table 3).

N S N a O O 1 O R 19a-f 1 Br S O O R 1 N S S 23a R = Ph, R = cPr R N 1 H 23b R = Ph, R = mCF3Ph O O N 23c R = 1-naphthyl, R1 = cPr O 23d R = 1-naphthyl, R1 = mCF Ph e O O 3 18a R1 = cPr O 1 18b R = mCF3Ph R1 O R1

b R S d R N S H N N

O O O O O O 1 1 20a R = N3, R = cPr 22a R = Ph, R = cPr 1 1 20b R = N3, R = mCF3Ph 22b R = Ph, R = mCF3Ph c 1 1 22c R = 1-naphthyl, R = cPr 21a R = NH2, R = cPr 1 1 22d R = 1-naphthyl, R = mCF3Ph 21b R = NH2, R = mCF3Ph

Table 3. SN2 diversification of the bromomethyl substituted scaffolds. Conditions: a) amine, DMF, rt, 30 min, 81-86%; b) NaN3, DMF, rt, 15 min (20a = 89%, 20b =

90%); c) Zn (s), NH4Cl, EtOH:H2O (3:1), rt, 20 min (21a = 83%, 21b = 81%); d) benzoyl chloride or 1-naphthoyl chloride, TEA, DCM, rt, 18 h 79-83%; e) benzenesulfonyl chloride or 1-naphthalenesulfonyl chloride, TEA, DCM, rt, 18 h 78- 81%. R1 Amine Yield (%) Product cPr N-piperidine 81 19a mCF3Ph N-piperidine 83 19b cPr N-morpholine 84 19c mCF3Ph N-morpholine 82 19d cPr N-1, 2, 3, 4-tetrahydroisoquinoline 84 19e mCF3Ph N-1, 2, 3, 4-tetrahydroisoquinoline 86 19f

The bromomethyl substituted ring-fused compounds 18a and 18b were also reacted with phenolic nuchleophiles. In this case, the potential for transesterification had to be considered when deciding upon base/solvent reaction conditions. Performing the reaction in DMF and Cs2CO3 at 0 °C gave the desired compounds in 82-96% yields with no transesterification observed (Scheme 8).

R1 R1 S R Br a O S N N

O O O O O O 1 18a R1 = cPr 24a R = Ph, R = cPr 1 24b R = Ph, R1 = mCF Ph 18b R = mCF3Ph 3 24c R = 1-naphthyl, R1 = cPr 1 24d R = 1-naphthyl, R = mCF3Ph

Scheme 8. SN2 functionalization with phenols. Conditions: a) phenol or 1-naphthol,

DMF, Cs2CO3, 3Å MS, 0 oC, 3 h (24a, b, c, d = 96%, 82%, 94%, 84%).

To further increase the molecular diversity accessible from this bromomethyl substituted 2-pyridone scaffold, we investigated making C-C bonds via the Suzuki⎯Miyaura coupling. Applying the previously used ligand free 52, 60 conditions (R-B(OH)2 Pd(OAc)2 and KF in MeOH), gave only 11% conversion to the desired product (Table 4). Consequently a catalyst screen with different Pd-sources was performed (Table 4).

F F F F OH F F HO B

Br S a S N N O O O O O O O O 26 18b 25

Table 4. Pd-screen in the Suzuki⎯Miyaura coupling, conditions a) 26, ”Pd-source”, KF, MeOH, MWI 110 oC for 10 min ”Pd-source” Amount (mol%) Yield

Pd(OAc)2 10 11 Pd(PPh3)2Cl2 5 84 Pd-NHC 5 50

Pd(PPh3)4 5 65

Pd(PPh3)2Cl2 was found to be the best catalyst for this reaction in this screen, and moreover the catalyst loading could be reduced to 5 mol%. Applying these conditions, a set of boronic acids were coupled with the bromomethyl substituted 2-pyridones 18a,b in 55-88% yields (Table 5).

1 R R1 S Br a R S N N

O O O O O O 18a R1 = cPr 27a-g 1 18b R = mCF3Ph Table 5. Suzuki⎯Miyaura couplings of the bromomethyl substituted scaffolds 18a- b. Conditions: a) R-B(OH)2, Pd(PPh3)2Cl2, KF, MeOH, MWI 110 oC for 10 min. R1 R Yield (%) Product cPr 72 27a

O cPr 77 27b

mCF3Ph N 88 27c H cPr F 56 27da O a mCF3Ph 61 27e

cPr 55 27fa a mCF3Ph 58 27g O aPurified by HPLC

The synthesized esters 19a-f, 22a-d, 23a-d, 25 and 27a-g were hydrolyzed and tested for their ability to inhibit biofilm formation. A 60% inhibition cut off at 200 µM concentration was set as a limit for ”biologically interesting” compounds. Compounds that fulfilled that criteria were further serially diluted and tested at different concentrations to generate EC50 values. The results are summarized in Table 6.

R1 R1 S S R a R N N

O O O OR2 O O 28a-z Table 6. Hydrolysis and biological evaluation, conditions a) LiOH, THF, 4-24 h 1 2 Ester Product R R R 200 µM EC50 (µM)

19a 28a N cPr Li NAa -

19b 28b mCF3Ph Li 30% -

19c 28c N cPr Li NAa - O a 19d 28d mCF3Ph Li NA -

19e 28e N cPr Li NAa -

19f 28f mCF3Ph Li 84% 130 O 22a 28g cPr H NAa - N H a 22b 28h mCF3Ph H NA - 22c 28i O cPr H NAa - N 22d 28j H mCF3Ph H 65% 130 O O a 23a 28k S cPr H NA - N H 23b 28l mCF3Ph H 48% O O a 23c 28m S cPr H NA - N 23d 28n H mCF3Ph H 91% 38 24a 28o cPr H 43% - O 24b 28p mCF3Ph H 80% 21 24c 28q cPr H 96% 32

24d 28r mCF3Ph H 72% 37 O 27a 28s cPr H NAa -

25 28t O mCF3Ph H 93% 9 27b 28u cPr H 37% - N 27c 28v H mCF3Ph H 97% 30

27d 28w F cPr H 76% 43 b 27e 28x O mCF3Ph H 75% 3

27f 28y cPr H - 1.4c c 27g 28z mCF3Ph H - 1.9 O

- 29a cPr H 54% 189

- 29b mCF3Ph H 92% 17 aNot active, bNever reached higher inhibition than 80% even at 200 µM cBiofilm inhibition data published in ref 43.

In conclusion, we have developed synthetic pathways to the bromomethyl substituted ring-fused 2-pyridone scaffolds 18. These compounds have been further evaluated in SN2 reactions with nitrogen and oxygen based nucleophiles. Furthermore C-C bond formation via the Suzuki⎯Miyaura coupling also proved effective on these scaffolds. In total 26 compounds were constructed via this route, hydrolyzed and evaluated for their ability to inhibit pili dependent biofilm formation. While many of the compounds showed low or no activity, the best compounds had EC50 values in the low µM range (Table 6, compounds 28t, x, y, z). The developed bromomethyl scaffolds have also been used in the synthesis of coumarin labeled compounds that will be introduced in Paper III. In addition this type of halo- methyl substituted scaffolds (i.e chloromethyl) have proved useful as key intermediates in the synthesis of 5- to 10 membered ring-fused tricyclic 2- pyridones.61

Fluorescence: lighting up bacterial virulence (Paper III)

In order to further study bacterial virulence mechanisms in uropathogenic Escherichia coli (UPEC), a biologically traceable label such as a fluorescent tag was desirable. The normal approach for labeling with fluorescent tags is to attach the fluorophore to the molecule via a linker. Considering the low molecular weight of our compounds, this approach would result in a large change of the molecule and potentially decrease the activity and bioavailability of these compounds. A more suitable approach in this case would be to exchange an existing moiety with a structural similar fluorescent tag to hopefully retain biological activity. We became interested in the coumarin and BODIPY fluorophores (Figure 8) because of their relatively small size, spectral properties, photostability, lipophilicity and lack of net ionic charge.62 It has previously been shown that a larger lipophilic group in positions 7 and 8 (Figure 8) of thiazolino ring-fused 2-pyridones is advantageous for their antibacterial properties.33e, 34b, 39, 43 Accordingly these positions were selected for the fluorophore attachment. The synthesized compounds were evaluated for their ability to inhibit the chaperone usher pathway, as well as for their capability to inhibit the CsgA polymerization by the previously described assays. In addition their photophysical and fluorescent properties [i.e quantum yields (ΦF)] were also recorded.

5 4 1 8 7 6 3 2 6 2 7 N 4 N O O 3 B 5 8 1 F F Coumarin BODIPY

Figure 8. General structures and numbering of the coumarin and 4, 4-difluoro-4- bora-3a, 4a-diaza-s-indacene (BODIPY) fluorophores.

Synthesis of the coumarin analogues

To best mimic the geometry of the naphthyl group, substitution through the 4-position of coumarin was prefered (Figure 8), with an additional electron donating substituent in the 7-position of the coumarin desirable to enhance fluorescence properties. The synthesis of the coumarin functionalized ring- fused 2-pyridones started from the commercially available 7-methoxy coumarin-4-yl acetic acid 30. Treating this together with Meldrum’s acid, DCC and DMAP gave acyl Meldrum’s acid derivative 31 in 62% yield. The

following acid induced ketene/imine cyclocondensation with the 2- thiazolines 17 and 32 furnished the ring-fused 2-pyridone esters 33 in 68- 86% yields. Treatment of esters 33a-d with LiOH in THF:MeOH (4:1) followed by acidic workup gave the corresponding carboxylic acids 34a-d in 53-66% yields (Scheme 9).

O O O OH

O O O a HO b O O O O O O O 30 O O 31

O R1 S R1 O N 1 S 17a R = cPr O O OR 17b R1 = mCF Ph O 3 N 32a R1 = Ph 1 1 33a R = Me, R = Ph O 32b R = 2-thienyl 1 O 33b R = Me, R = mCF3Ph 33c R = Me, R1 = 2-thienyl c 33d R = Me, R1 = cPr 34a-d R = H

Scheme 9. Synthesis of analogues functionalized with coumarin in the 7-position. Conditions: a) DCC, DMAP, DMF, rt 16 h, 62%; b) 17 or 32, TFA, DCE, MWI 120 oC 140 s (33a-d: 66, 58, 53 and 66% respectively); c) i) 0.1M LiOH (aq), THF:MeOH 4:1 ii) H+ (34a-d: 66, 58, 53 and 66% respectively).

Compounds 34a-d exhibited relatively low quantum yield values (ΦF) of 5, 1, 0.7 and 0.4% respectively. This phenomenon was thought to result from quenching of the fluorophore due to the close proximity of the central 2- pyridone. To investigate this a second set of compounds were synthesized where the spacer between the central fragment and the fluorophore was extended by a methylene. Deprotonated 4-methyl coumarins 35a-b were used as nucleophiles with the bromomethyl substituted 2-pyridones 18a-b,63 affording both the methoxy (36a-b) and the diethylamine (36c-d) substituted coumarin analogues in yields of 74-83% (Scheme 10).

R1 O R1

Br S a S b N N R O O O O O O 1 18a R1 = cPr 36a R = cPr, R = MeO- 1 18b R1 = mCF Ph 36b R = mCF3Ph, R = MeO- 3 1 36c R = cPr, R = Et2N- 1 36d R = mCF3Ph, R = Et2N-

O R1 S N R O O R O OH O 35a R = MeO- 1 2 37a R = H, R = cPr, R = MeO- 35b R = Et2N- 1 2 37b R = H, R = mCF3Ph, R = MeO- 1 2 37c R = H, R = cPr, R = Et2N- 1 2 37d R = H, R = mCF3Ph, R = Et2N-

Scheme 10. Synthesis of analogues functionalized with coumarin in the 7-position with extended linkers. Conditions: a) 35a or 35b, LiHMDS, THF, -35 °C 1 h (36a-d: 82, 74, 75 and 83% respectively); b) For 37b,d i) 0.1M LiOH (aq), THF:MeOH 4:1,

1.5h, ii) H+ (37b = 86%, 37d = 82%); For 37a,c i) LiBr, TEA, MeCN (2% v/v H2O), rt, 3 h, ii) H+ (37a = 91%, 37c = 81%).

The following hydrolysis proved problematic for the cyclopropyl substituted coumarin analogues 36a and 36c under the standard conditions (LiOH, THF). This problem was circumvented by using a hydrolysis method previously reported for α-hetroatom substituted esters,64 and with both these hydrolysis methods the desired carboxylic acids 37a-d were isolated in 81- 91% yields. Unfortunately extension to an ethyl linker had no affect on the quantum yields. However, replacing the methoxy substituent in the 7- position of the coumarin with a diethylamine substituent gave increased quantum yields and compounds with acceptable fluorescent properties.

R 1 n R S O N

O O OH O Table 7. Photophysical properties and biological evaluation of analogues containing coumarin fluorophores in the 7-position 1 a a b,c b,d b ID R R n EC50 EC50 λabs λfl ΦF [%] Pili Curli [nm] [nm] (λex nm) [µM] [µM] 34a MeO- Ph 1 >200 NAe 328 394 5 (330)f e f 34b MeO- mCF3Ph 1 >200 NA 330 420 1 (330) 34c MeO- 2-thienyl 1 >200 NAe 328 396 0.7 (330)f 34d MeO- cPr 1 >200 NAe 328 413 0.4 (330)f 37a MeO- cPr 2 156 NAe 327 393 0.5 (330)f

37b MeO- mCF3Ph 2 65 175 329 430 0.6 (355)g e f 37c Et2N- cPr 2 12 NA 392 484 6 (330) 37d Et2N- mCF3Ph 2 18 25 393 474 15 (390)f aestimated from 16-32 data points for every concentration, bAll substances were dissolved in DMSO and subsequently diluted in phosphate buffer at pH 7.0. The samples DMSO concentrations never exceed 5 wt %. The sample concentration in the DMSO stock solutions are adjusted so that the final samples never have peak absorbance higher than 0.1, cWavelengths of the peak absorption, dThe peak fluorescence, eNot active, fReference: POPOP in MeOH, gReference: Perylene in cyclohexane.

Consequently the diethylamine substituted coumarin was introduced in the 8-position of the ring-fused 2-pyridone. Starting by deprotonation of 35b by LiHMDS in THF and subsequent quenching with ethyl 2-bromoacetate gave sluggish reactions with poor conversions. By adding DMPU to the reaction mixture the desired coumarin ester 38 could be isolated in 77% yield. LiOH hydrolysis of 38 followed by conversion to the corresponding acid chloride, and subsequent treatment with L-cysteine methyl ester hydrochloride salt in the presence of TEA gave complex reaction mixtures. Unfortunately, even a HBTU mediated coupling with the carboxylic acid failed to give the desired product. Exchanging L-cysteine methyl ester for S-trityl-L-cysteine methyl ester (39) together with HBTU in DMF gave compound 40 in 60% yield over

2 steps. Direct ring-closure of the S-trityl protected cysteine amide with TiCl4

was possible following a reported procedure,65 and the desired 2-thiazoline 41 was obtained in 40% yield. The ketene/imine cyclocondensation proceeded well and the ring-fused 2-pyridone ester 43 was isolated in 73% yield. The following hydrolysis of 43 with LiOH was effective and gave the final product 44 in 91% yield (Scheme 11).

Et2N O O Et2N O O Et N O O 2 a b

40 HN O EtO O 35b MeO STrt 38 O

NEt2 NEt2

O O O c d O

S O S N N

41 O O O O O R 43 R = Me e 44 R = H

O O O TrtS O O NH 2 HO O 39 O 42

Scheme 11. Synthesis of the 8-position coumarin functionalized compound 44.

Conditions: a) HMDS, n-BuLi, DMPU, BrCH2COOEt, THF, -20 oC, 35 min, 77%; b) i)

1M LiOH (aq), THF, rt, 16 h ii) HBTU, 39, DMF, rt, 17 h, 60% c) TiCl4, DCM, 0 oC to rt, 16h, 40% d) 42, TFA, DCE, MWI 120 oC, 3 min, 73%; e) i) 0.1M LiOH (aq), THF, rt, 1 h, ii) AcOH, 91%. Compound 39 was synthesized from the corresponding commercially available carboxylic acid by treatment with TMS-diazomethane in DCM:MeOH 9:1 and used without purification.

Synthesis of the BODIPY analogues

To further increase the possibility of finding a bioactive compound with useful fluorescent properties, the corresponding ring-fused 2-pyridone with the BODIPY fluorophore in the 8-position was synthesized (Figure 8). The known BODIPY containing carboxylic acid 4566 was first synthesized from succinic anhydride and 2,4-dimethylpyrrole under microwave irradiation (Scheme 12). Conversion of 45 to the corresponding acid chloride followed by addition of L-cysteine methyl ester hydrochloride salt and TEA gave the 65 cysteine amide 46 in 64% yield. The TiCl4 induced ring-closure was followed by treatment with BF3*Et2O, due to exchange of the BF2 moiety in BODIPY by TiCl2 during the condensation reaction, to furnish the 2- thiazoline 47 in 75% yield. The acid induced ketene/imine cyclocondensation reaction proceeded well and gave 48 in 64% yield. The hydrolysis of compound 48 proved to be very demanding, requiring extensive screening of hydrolysis, protocols, of which LiI in pyridine under microwave irradiation was found to be the best. Subsequent treatment by

BF3*Et2O was again necessary to reinstall the BF2 moiety which was lost during the hydrolysis procedure, giving 49 as a racemate in 29% yield.

F F B F F N N B N N a b c O O O

HN O HO O O SH

45 O 46

F F F N B F B N N N d 48 R = Me S S e O 49 R = H N N

O O O 47 O O R Scheme 12. Synthesis of the 8-position BODIPY functionalized compound 49.

Conditions: a) 2,4-dimethylpyrrole, BF3*Et2O, TEA, DCE, MWI 120 oC for 3x60 min,

16%; b) i) (COCl)2, DCM, rt, 17 h, ii) L-cysteine methyl ester hydrochloride, TEA,

DCM, 0 oC to rt, 4.5 h, 64%; c) i) TiCl4, DCM, 0 oC to rt, 4.5 h, ii) BF3*Et2O, DCM, rt, 40 min, 75%; d) 42, TFA, DCE, MWI 120 oC for 140 s, 64% e) i) LiI, pyridine, MWI

140 oC for 15 min, ii) TEA, BF3*Et2O, DCE, 80 oC, 15 min, 29%.

Evaluation of the photophysical properties of 44 and 49 revealed that the quantum yield of 49 was surprisingly low for a BODIPY-derivative (ΦF = 10%, Table 8). In this case an intramolecular photo quenching by the neighboring ring-fused 2-pyridone was believed to be the problem. We reasoned that a rigid phenyl spacer between the 2-pyridone core and the BODIPY moiety might solve the problem. This would not only put the BODIPY moiety further away from the 2-pyridone core, but also restrict its rotation which is beneficial for generating higher quantum yields. The synthesis of this compound started from the known carboxylic acid 9b.52 Conversion of the carboxylic acid to the corresponding acid chloride and subsequent treatment with 2,4-dimethylpyrrole, BF3*Et2O and TEA gave the BODIPY functionalized compound 50 in 15% yield (Scheme 13). Hydrolysis under standard conditions followed by acidic workup, gave the carboxylic acid 51 in 84% yield.

F F B N N O OH

a S S N N

O O O O O O 9b R 50 R = Me b 51 R = H Scheme 13. Synthesis of the phenyl spacer analogue 51. Conditions: a) i) (COCl)2,

DMF, DCM, rt, 1 h ii) TEA, BF3*Et2O, 2,4-dimethylpyrrole, DCE, MWI 140 oC for 50 min, 15%; b) i) 0.1 M LiOH (aq), THF, rt, 1h ii) H+, 84%.

To our delight the quantum yield of this compound increased dramatically to 67% (Table 8). In addition the biological efficacy of this compound against both pili and curli was in the low µM range (Table 8).

F F B NEt2 N N F F N B O N

O S O S S N N N

O OH O OH O OH O O O 44 49 51 Table 8. Photophysical and biological evaluation of compounds 44, 49 and 51 a a b,c b,d b ID EC50 Pili EC50 Curli λabs [nm] λfl [nm] ΦF [%]

[µM] [µM] (λex nm) 44 5 17 396 478 11 (346)e 49 4 14 506 524 10 (470)f 51 14 12 502 514 67 (470)f aestimated from 16-32 data points for every concentration, bAll substances were dissolved in DMSO and subsequently diluted in phosphate buffer at pH 7.0. The samples DMSO concentrations never exceed 5 wt %. The sample concentration in the DMSO stock solutions are adjusted so that the final samples never have peak absorbance higher than 0.1, cWavelengths of the peak absorption, dThe peak fluorescence, eReference: POPOP in Me OH, fReference: Rhodamine 6G in water,

These intriguing results encouraged us to also investigate BODIPY attachments in the 7-position of the ring-fused 2-pyridones. First, the carboxylic acid 45 was converted to the corresponding acyl Meldrum’s acid derivative 52 under standard conditions in 82% yield. The ketene/imine cyclocondensation followed by LiOH hydrolysis in THF:MeOH 4:1 gave compound 54 in 88% yield. Once again the quantum yield was disappointingly low for a BODIPY containing compound (ΦF = 11%, Table 9). To investigate the possible photoquenching of BODIPY by the thiazolino sulfur atom, the corresponding oxazolino analogue 57 was made. The oxazolino analogue 57 was synthesized in 73 % yield via a 2 step, one pot procedure with serine amide 55 according to a previously developed protocol.67 A consequent LiBr/TEA mediated hydrolysis in wet MeCN gave 57 in 88% yield.64 The ~3-fold increase in quantum yield indicated that the sulfur was involved in photoquenching. To investigate this further, the sulfoxide analogue 58 was made from the corresponding sulfide (54) by oxidation with mCPBA in DCM at rt in 80% yield. Photophysical investigation of 58 revealed a substantial ~7-fold increase in quantum yield (Table 9).

F F F F N B B N F N N N B a b F N S HO O N

HO O O O O O O O R 45 53 R = Me c 52 54 R = H Ph

S d e N

O O F 32a N F B N F F B O OH N O O N S Ph OMe N N N H O O O O OH 55 O R O 56 R = Me 58 f 57 R = H Table 9. Synthesis and evaluation of the BODIPY analogues. Conditions: a) Meldrum’s acid, DCC, DMAP, DCM, 0 oC to rt, 8h, 82%; b) 32a, TFA, DCE, MWI 120 oC for 140 s, 85%; c) i) 0.1 M LiOH (aq), THF:MeOH 4:1, 88%, ii) AcOH, 88%; d) 55,

(NH4)2MoO4, toluene, reflux, soxhlet (3Å MS), 2 h, 73%; e) Starting from 54: mCPBA, DCM, rt, 15 min, 80%; f) i) LiBr, TEA, MeCN (2 v/v % H2O), rt, 3 h ii) AcOH, 88%.

a a b,c b,d b ID EC50 Pili EC50 Curli λabs [nm] λfl [nm] ΦF [%] [µM] [µM] (λex nm) 54 10 24 498 509 11 (480)e 57 13 14 497 531 27 (480)e 58 29 40 498 516 71 (480)e aestimated from 16-32 data points for every concentration, bAll substances were dissolved in DMSO and subsequently diluted in phosphate buffer at pH 7.0. The samples DMSO concentrations never exceed 5 wt %. The sample concentration in the DMSO stock solutions are adjusted so that the final samples never have peak absorbance higher than 0.1, cWavelengths of the peak absorption, dThe peak fluorescence, eReference: Rhodamine 6G in water

The strategy of exchanging an existing group for a fluorophore of similar size successfully generated compounds with good biological activity against both pili and curli and good fluorescent properties. The best fluorophore containing compounds prepared were the position 8 substituted ring-fused

2-pyridones 44 (coumarin) and 49 (BODIPY), with pili formation EC50 = 5 and 4 µM respectively. They also demonstrated good inhibitory effect against curli formation (44: EC50 = 17 µM; 49: EC50 = 14 µM). These EC50 values can be compared with the best published compound called FN075 (29b) with reported EC50 values against formation of pili and curli of 17 and 38 µM respectively.68 Compounds 49 and 54 subsequently underwent further evaluation in a bacterial population, in which the UPEC strain UTI89 was grown under pili dependent conditions in the presence of 49 or 54, and the results examined by fluorescence microscopy (Figure 9). It is known that even under pilus-inducing conditions not all bacteria in a broth actively assemble pili. We can conclude from fluorescence microscopy images that our compounds indeed bind somewhere to the pili assembly machinery and not randomly throughout the bacteria. Furthermore, it appears 49 is enriched in the outer part of the bacteria, which may indicate enrichment in the periplasm.

Figure 9. Fluorescence microscopy images of bacteria treated with fluorescent 2- pyridone analogues 49 or 54 respectively.

Triazoles (Paper IV)

The triazoles are interesting heterocycles in the area of medicinal chemistry and probably most well known as peptide bond mimetics69 (Figure 10A) They have aslo shown interesting characteristics as double bond70 (Figure 10B and 10C) and furan71 mimetics.

2 3 O N N 3 N 1 R1 N R1 N N 4 N 2 R1 N R1 R N R 4 R R1 5 N 1 5 1 H R R R R A B C

Figure 10. Triazoles as mimetics for A) amide bonds, B) E-olefins and C) Z-olefins

The synthesis of triazoles through the thermal 1,3-dipolar cycloaddition between an alkyne and an azide was first developed by Rolf Huisgen in the beginning of the sixties.72 In recent years K. Barry Sharpless, Morten Meldal and Valery V. Fokin have developed triazole chemistry further to allow selective synthesis of the 1,4- or 1,5-regioisomers (Figure 10B and 10C) by introducing copper(I)73 or ruthenium(I)74 catalysts. These reactions are today commonly referred to as the copper(I) catalyzed azide alkyne cycloaddition (CuAAC) or ”click reaction”, and ruthenium(I) catalyzed azide alkyne cycloaddition (RuAAC). The CuAAC reaction is today applied across a broad variety of areas such as drug discovery, materials science and chemical biology.75 Ring-fused 2-pyridones with heterocyclic substituents in the 2- and 8-position (i.e. thiophenes and furanes) have previously been synthesized and showed promising biological activities against pili formation in the pili dependent biofilm assay (Figure 11).43, 60

S X S N N

O OH O OH O O

59a X = O, EC50 = 78 µM 59b X = S, EC50 = 25 µM 60 EC50 = 39 µM Figure 11. Previously synthesized heterocycle substituted ring-fused 2-pyridones.

The intriguing biological results of these compounds encourged us to investigate what impact other heterocycles would have on their biological activity against pili formation. We directed our attention towards triazoles for two main reasons: Pd-catalyzed cross coupling reactions in position 2

and 8 had previously proven successful, and the CuAAC reaction provided easy access to structurally diverse triazoles.37, 52, 60, 76 With this in mind, we envisioned a synthetic pathway starting from the known halogenated compounds 6 and 61 (Figure 12).37, 52

Sonogashira R coupling N N CuAAC N reaction I S S Hydrolysis N N H Br O OH O O Transfer O O hydrogenation 6

Sonogashira coupling R1 R1

S N N S Br N N N R O OH O O O CuAAC O reaction 61a R1 = cPr Hydrolysis 1 61b R = mCF3Ph Figure 12. Retrosynthetic analysis of triazole functionalized ring-fused 2-pyridones.

Functionalization of the 8-position The functionalization of the 8-position was straightforward and began with a Sonogashira coupling on the known bromo-iodo ring fused 2-pyridone 6,52 and subsequent TMS deprotection with K2CO3 in MeOH:THF (4:1) to give 62 in 76% yield. The terminal acetylene 62 was then exposed to an CuAAC reaction in 1:1 DMF:H2O with a variety of azides in yields of 42-95% (Table 10).

R N N N I S a S b S N N N Br Br Br O O O O O O O O O 6 62 63a-i Table 10. Triazole functionalization of the 8-position. Conditions: a) i)

Pd(PPh3)2)Cl2, CuI, TMSA, TEA, DMF, rt, 20 h, ii) K2CO3, MeOH:THF 4:1, rt, 15 min,

76%; b) CuSO4, Na-ascorbate, NaN3, R-X, DMF:H2O 1:1, 70 oC, 16 h. Product R X Yield (%) a 63a H N3 42 63b Me I 79 63c Et I 78

63d cPrCH2 Br 54 63e Bn Br 70 63f pMeBn Br 82

63g pNO2Bn Br 84 OH b 63h N3 66

OAc 63i AcO OAc N3 95

O aTMSN3 was used instead of NaN3; bThe azido alcohol was synthesized from the corresponding amino alcohol with TfN3 according to published procedures.77

The bromo esters 63a-i were hydrolyzed under standard conditions at this stage in order to test these interesting intermediates for their ability to inhibit the pili assembly machinery. The resultant carboxylic acids 64a-i were further reacted with ammoniumformate and Pd/C in refluxing MeOH to give dehalogenated compounds 65a-i in 67-92% yields (Table 11).

R R N N N N N N

S b S N N Br O O O OH O O R1 63a-i R1 = Me 65a-i a 64a-i R1 = H Table 11. Hydrolysis and biological evaluation of the 8-position triazole

functionalized compounds, conditions a) 1.0 M LiOH (aq), THF, rt, 17 h b) HCO2NH4, Pd/C, MeOH, reflux, 4 h

R Product Yield EC50 Product Yield EC50 (%) (µM) (%) (µM) H 64a 72 >200 65a 73 >200 Me 64b 73 >200 65b 85 >200 Et 64c 87 >200 65c 69 >200

cPrCH2 64d 93 >200 65d 72 >200 Bn 64e 85 100 65e 67 100 pMeBn 64f 82 200 65f 92 75 a pNO2Bn 64g 63 35 65g 82 >200 OH 64h 77 150 65h 88 >200

OH HO OH 64i 79 >200 65i 74 >200

O aThe nitro group was reduced to the corresponding aniline

Unfortunately none of the 18 synthesized compounds demonstrated noteworthy biological activity in the pili dependent biofilm assay, except 64g

(EC50 = 35 µM) which showed comparable activity to the previously synthesized thiophene substituted 2-pyridone 60 (EC50 = 39 µM; Figure 9).

Functionalization of the 2-position The first attempt to functionalize position 2 with a triazole was made with the known alkyne substituted methyl ester 66.76 The following CuAAC

reaction in DMSO:H2O 1:1 proceeded well and the corresponding methyl triazole 67 was isolated in 71% yield (Scheme 14). However the following methyl ester hydrolysis turned out to be very problematic, with only the decarboxylated 68 isolated (Scheme 14). Previously we developed hydrolysis methods for other 2-position heterocycle substituted compounds (e.g 59, Figure 10) but unfortunately even this method (25 eq KOH, THF:MeOH 2:1, MWI 90°C 25 min) failed together with the triazole substituent.

S a S N N N N N

O O O O O O 67 66

b or c or d S N N N N

O 68 Scheme 14. Attempted hydrolysis of the triazole substituted ring-fused 2-pyridone.

Conditions: a) CuSO4, Na-ascorbate, NaN3, MeI, DMSO:H2O 1:1, 80 oC 1h, 71%; b) LiOH, THF, rt, 18 h; c) LiI, pyridine, MWI 140 °C 30 min; d) KOH, THF:MeOH 2:1, MWI 90 °C 25 min

To solve this problem, we required an ester that can be deprotected under milder conditions, whilst still being resilient enough to withstand the conditions used in the synthesis of 67. The previously developed trimethylsilyl ethyl (TMSE) ester was used for this purpose.76 After some modifications of the solvent due to the higher lipophilicity of these esters, the CuAAC reaction was successful and to our delight the following TBAF deprotection of TMSE esters 69a-b could be performed on the crude material. The final triazole substituted ring-fused 2-pyridones 70a-f were isolated in 54-69% yields over 2 steps without any decarboxylation or 1,5- substituted triazole regioisomer detected. Compounds 70a-f were evaluated for their ability to inhibit pili assembly in UPEC (Table 12).

R1 R1 S a S N N N N N R O O O OH O O

69a R cPr Si 70a-f 69b R = mCF3Ph Table 12. Triazole functionalization of the 2-position. Conditions: a) i) CuSO4, Na- ascorbate or CuTC, R-X, NaN3, DMSO:H2O 9:1, for 70a-c 18 h at rt, for 70d-f 2 h at

60oC, ii) TBAF*3H2O, THF, rt, 3 h. 1 Compound R R X Yield (%) EC50 (µM) 70a cPr H 56 75 70ba cPr Me I 69 150 70cb cPr Bn Br 54 >200

70d mCF3Ph H 61 >200 c,e 70e mCF3Ph Me I 61 9 d 70f mCF3Ph Bn Br 55 50 aApprox. 5% of 70a was formed, bApprox 10% of 70a was formed cApprox 5% of 70d was formed, dApprox. 10% of 70d was formed, eCuTC was used instead of CuSO4/Na- ascorbate.

In conclusion, we have developed synthetic methods for functionalizing the ring-fused 2-pyridones with triazoles in position 2 and 8. This was effected via a CuAAC reaction onto the corresponding terminal acetylene with complete regioselectivity for 1,4-substituted triazoles. In the case of the 2- position functionalization (70), the TMSE ester was used due to problems with the hydrolysis of the corresponding methyl ester. The TMSE ester was effectively removed in situ by TBAF in THF at rt, without any decarboxylation detected. Furthermore, all the twenty-four synthesized compounds were evaluated for their ability to inhibit pili assembly in the pili dependent biofilm assay. Most compounds in this collection showed low or no activity, except for 70e (EC50 = 9 µM; Table 12). These results demonstrate that position 2 is an interesting position for further exploration.

Acetylene spacer analogues (Paper V)

Compound 29b (also known as FN075) has shown interesting biological properties in both α-synuclein fibrillation78 and in inhibition of curli formation.39a To further study the ”chemical space” of this compound a large set of molecules exhibiting structural resemblance to 29b was synthesized and screened. As previously mentioned the carboxylic acid is pivotal for biological activity. Furthermore, previous results indicated that a large aryl substituent in position 7 and an electron deficient aryl or heteroaryl in position 8 (Figure 13) was useful to obtain biological activity against curli formation.39a, 68

Electron deficient aryl or heteroaryl preferred

CF 3 Site available for transformation

Large aromatic group preferred S N Vital for O OH biological O activity

Site available for transformation

Figure 13. Previously determined structure activity of FN075.

Taking 29b as the starting point, and considering the preferred moieties (Figure 13), a set of analogues exploring the available positions was synthesized according to previously developed routes (Figure 13). Firstly, the sulfur substituent in the thiazolino ring was either removed78a or oxidized to the corresponding sulfoxide78a/sulfone79 (Figure 14, routes A and B,). Secondly, position 6 in the pyridone ring was functionalized with an amine or ring-fused as a pyrazole with the benzylic CH2 bridge in position 7 to form an extended peptidomimetic backbone (Figure 14, routes C and D,).38 Thirdly, the thiazolino ring was oxidized and expanded by one nitrogen to form the corresponding six membered sultams (Figure 14, route E).79

CF3 CF3

X N N O OH O O OH O X = SO B A X = SO2

CF3

CF 8 1 3 7 S CF3 C 2 E 6 N 3 5 4 O O X O O O S R N N H2N N O OH O D O X = S O OH X = SO2 R = H R = Me CF3

S N N N H O OH O

Figure 14. Transformations made to the known inhibitor of curli formation FN075.

To investigate the impact the sterics and positioning of the substituent in position 8 had on the biological activity, a series of analogues containing an acetylene spacer were synthesized via the Sonogashira coupling onto the bromo-iodo compound 6 (Table 13).52 The strategy to include a rigid linker between the pyridone core and the position 8 substituent had previously successfully been used in analogues containing the BODIPY fluorophore with retained biological activity (i.e 51, Table 8).68 In addition, this strategy could also give access to triazole substituted compounds via the CuAAC reaction with the terminal acetylene, as presented in Paper IV.55

R R

S a S c S N N N Br Br O O O O O OH O O 1 O R 73a-c 6 71a-c R1 = Me b 72a-c R1 = H

Table 13. Synthesis of analogues containing an acetylene spacer. Conditions: a)

Pd(PPh3)2Cl2, alkyne, CuI, TEA, DMF, rt or 50 oC; b) LiOH in THF at rt or LiI in pyridine, MWI 130 oC, 15 min; c) Zn(s), AcOH, 100 oC.

Entry R Compound Compound Compound (yield %) (yield %) (yield %)

1 cPr 71a (82) 72a (63) 73a (75)

2 Ph 71b (82) 72b (67) 73b (83)

a b 3 mCF3Ph 71c (91) 72c (75) 73c (63) aReaction performed at 50 °C, bThe LiI/pyridine method was used

The hydrolysis of the mCF3Ph substituted 71c was slow and never reached completion under standard hydrolysis conditions (LiOH, THF), however this problem could be ameliorated by switching to LiI in pyridine and microwave irradiation. The bromine substituted intermediates were hydrolysed at this stage to test the activity of these intermediates (72a-c). Removal of the bromine in position 6 with Zn(s) in AcOH also gave compounds 73a-c in yields of 63-83%. The ammoniumformate, Pd/C system previously used for the triazole substituted compounds (64a-i, Table 11) could not be employed in this case due to the sensitivity of the alkyne under these conditions. A library constituting approximately 100 compounds previously synthesized with changes to the FN075 core structure together with the acetylene spacer analogues was screened to examine in the thioflavin T (ThT) in vitro assay for their ability to inhibit CsgA polymerization into a functional amyloid fibre.38, 55, 78a, 79 ThT is known to bind to amyloid fibres, and when that occurs the intensity of ThT fluorescence increases. This increase in fluorescence is proportional to the amount of amyloid fibres formed and is measured by a photospectrometer. All experiments were performed twice in duplicates. Two compounds excelled amongst those screened: 72b,c which both contained an acetylene spacer in position 8 and a bromine substituent in position 6 of the 2-pyridone ring. They acted as

accelerators of CsgA polymerization into a functional amyloid fibre (Figure 15) and accelerated formation of mature amyloid fibres within 6 h, a process that normally takes at least 18 h. To the best of our knowledge, there are no such accelerators previously reported in literature. To determine the relative importance of the acetylene spacer and bromine substituent on the observed templating effect, the brominated analogue of FN075 (76, Scheme 15) was synthesized. Methyl ester 74 was first brominated in position 6 with NBS in acetonitrile at rt in excellent yield, followed by a standard LiOH hydrolysis in THF to give the brominated FN075 analogue 76 in 89% yield (Scheme 15).

F F F F F F F F F

S a S b S N N N Br Br O O O O O OH O O O 74 75 76

Scheme 15. Synthesis of the brominated analogue of FN075. Conditions: a) NBS, MeCN, rt, 91%; b) 1 M LiOH, THF, rt, 89%.

Compound 76 displayed no accelerating effect, instead acting as an inhibitor in the same way as 73b and 73c. These results verify that the combination of an acetylene spacer in position 8 and a bromine substituent in position 6 is required for the accelerating effect (Figure 15). The bromine substituent in position 6 is interesting for further explorations and replacing it for other halogens/groups will be investigated in future.

Figure 15. CsgA polymerization assay of compounds 72a-c, 73a-c and 76. ThT fluorescence measured over time at a compound concentration of 50 µM and a protein concentration of 5 µM

A set of 18 compounds from the initial library was further evaluated in the pellicle assay. The most active was 73c, which fully inhibited the formation of the pellicle biofilm at 3 µM. However 72b and 72c, which displayed accelerating effects in the ThT assay, did not affect pellicle biofilm formation. In summary approximately 100 modified compounds with starting point in FN075 were screened for their ability to inhibit CsgA polymerization via the ThT assay. The library screened contained both previously synthesized compounds with structural resemblance with FN075 according to Figure 14 as well as the acetylene spacer analogues presented in this chapter. The screen revealed clear structure activity relationships, with extension of the peptidomimetic backbone (Figure 14, routes C and D,) or the thiazolino ring expanded to a sultam (Figure 14, route E) resulted in more potent inhibitors. Strikingly analogues with an acetylene spacer were found to exhibit accelerating effects in the ThT assay. The combination of an acetylene spacer and a bromine substituent in position 6 of the 2-pyridone was required to achieve the accelerating effect. If either of these features was removed the compound instead acted as an inhibitor. These compounds are, as far as we know, the first reported accelerators of the CsgA polymerization process.

Asymmetric synthesis of Δ2-thiazolines (Paper VI)

Δ2-Thiazolines are common fragments represented in a variaty of compounds, among them natural products (e.g. Grassypeptolide A-C80 Pulicatin B81 and Micacocidin82), pheromones,83 and flavoring agents84 (Figure 16). They also possess intriguing properties as substituents in chiral ligands used in asymmetric synthesis85 (Figure 16). As asymmetric ligands they have shown promising results in a variety of reactions such as Diels- Alder reactions,86 Pd-catalyzed allylic substitutions,85a, 85b alkylzinc additions to aldehydes87 and Friedel-Craft alkylations.88 Furthermore Δ2-thiazolines can serve as interesting intermediates in organic- and biosynthesis.89

N N OH O O O O N S O HN * N O N S O HO HO HN O O S Pulicatin B N N NH R

Grassypeptolide A R = Et, * = R O Grassypeptolide B R = Me, * = R S S Grassypeptolide C R = Et, * = S N N SBT 2-acetyl-2-thiazoline

H S H H S OH N N S S N S OH N N N N S Ph2P HOOC R R R Thio-PyBox Thio-PHOX Micacocidin Figure 16. Examples of natural products (Grasspeptolide A-C, Pulicatin B and Micacocidin), pheromone (SBT = 2-sec-butyl-4,5-dihydrothiazole), flavoring agent (2-acetyl-2-thiazoline) and asymmetric ligands (Thio-PyBox and Thio-PHOX) containing the Δ2-thiazoline core.

As described earlier we are interested in Δ2-thiazolines as intermediates in an acylketene/imine cyclocondensation reaction for the construction of

thiazolino ring-fused 2-pyridones for use as antibacterial agents.39, 68 The most potent compound in the pili project so far display an EC50 of 0.4 µM in the pili-dependent biofilm assay previously described (78, Scheme 16).43 This compound initially was synthesized as a racemate via a conjugate addition by a higher order cuprate addition to the corresponding α,β- unsaturated methyl ester 77 (Scheme 16).37

S S

S a-b S EC50 = 0.4 µM N N

O O O OH O O 77 (±)-78 Scheme 16. Previous racemic synthetic route for the 2-phenyl substituted 78.

Conditions: a) Ph2CuCNLi2, THF, -78 oC, 20 min; b) LiOH, THF, rt, 16 h.

The potency of this compound encouraged us to develop asymmetric routes to 2-substituted ring-fused 2-pyridones. The previously used cuprate addition had proved problematic to scale, so development of an asymmetric version of that reaction was not an attractive option. Another possible route that we initially considered was the Sharpless asymmetric amino hydroxylation. However there are some drawbacks with that reaction, firstly it will give the syn-diastereomer of (±)-82 (scheme 18) when performed on commercially available trans-methyl cinnamate. Secondly that reaction is known to give mixtures of regioisomers and in some cases poor conversions.90 Looking through the literature, we noticed reports on the synthesis of 2,4,5-trisubstituted Δ2-thiazolines were scarce.91 To create robust, enantioselective and scalable synthetic pathways for future exploration, we directed our attention towards a synthetic pathway starting from the Sharpless asymmetric dihydroxylation of methyl cinnamate, a reaction known to proceed in good yields and high enantioselectivity.92 Sequential FGIs as illustrated in Scheme 17 would then provide the 5- substituted Δ2-thiazolines. The synthesized 5-substituted Δ2-thiazoline could then be used in the ketene/imine cyclocondensation reaction for the stereoselective synthesis of 2-substituted ring-fused 2-pyridones.

Deprotection followed by iminoether cyclocondenzation Mesylation followed by AcSK displacement R 1 2 SAc O OH O S 5 N O O 3 4 NHBoc OH O O 1) selective nosylation 2) NaN3 displacement 3) reduction/protection

Scheme 17. Retrosynthetic analysis to the 5-substituted Δ2-thiazolines

To enable development of the synthetic route, the reactions were first performed under racemic conditions. Commercially available methyl cinnamate was exposed to a K2OsO4*2H2O catalyzed dihydroxylation reaction using NMO as the stoichiometric oxidant to give diol (±)-79 in 81% yield. Conversion to the corresponding Boc-protected amino alcohol (±)-82 was made in analogy with published procedures for the corresponding ethyl ester93 in good yields via the nosylate ((±)-80) and azide ((±)-81, Scheme 18). The selectivity for the α-hydroxy in the nosylation reaction can be 94 attributed to the difference in pKa values between the hydroxyl groups. The Boc-protected amino alcohol (±)-82 was converted to the acyl protected amino thiol (±)-83 via a two-step reaction sequence in 60% yield. Ms2O was used instead of MsCl due to potential scrambling of the stereogenic center by the nucleophilic chloride ion.

OH O O OH O a b c O O O OH ONs (±)-79 (±)-80

OH O S O OH O f d O e O O HN O HN O N3 (±)-81 (±)-82 O (±)-83 O

S Cl NH N 2 O O O 85 (±)-84 Scheme 18. Synthesis of the thio amine (±)-83 and attempted cyclization.

Conditions: a) K2OsO4*2H2O (2 mol%), NMO, MeCN:acetone:H2O 1:1:1, rt, 18 h,

81%; b) NsCl, TEA, DCM, 0 °C, 1 h, 75%; c) NaN3, DMF 40 °C, 48 h, 70%; d) i)

SnCl2*2H2O, rt, 2 h; ii) NaHCO3, Boc2O, 1,4-dioxane:H2O, rt, 16 h, 93%; e) i) Ms2O,

TEA, DCM, 0 °C, 2 h; ii) KSAc, DMF, rt, 15 h, 60%; f) i) K2CO3 or NaOMe, MeOH, 2-5 h; ii) TFA or HCl, 2-5 h; iii) 85 , TEA, DCM, rt, 18 h.

Unfortunately all attempts to deprotect and ring-close (±)-83 together with 85 to yield the 5-substituted Δ2-thiazoline failed and gave complex reaction mixtures. We therefore envisioned synthesizing amido alcohols as alternate precursors for the Δ2-thiazoline synthesis (Scheme 19).95

Esterification O R OH O O O OH O

O O O HN N OH R 3 O 1) nosylation 2) NaN3 displacement N3 reduction followed by O-N acyl migration

Scheme 19. Retrosynthetic analysis of the amido alcohols.

Attempts to synthesize the amido alcohol (±)-87c from (±)-82 were made by deprotection of the Ac- and Boc-protective groups by NaOMe/MeOH or

K2CO3/MeOH and TFA/DCM or HCl/1,4-dioxane respectively followed by an amide coupling reaction. Unfortunately neither coupling by TBTU or acid

chloride worked. The esterification of azido alcohol (±)-81 was therefore investigated under different conditions: TBTU, acid chloride/TEA or DCC/DMAP (Steglich conditions),96 with the Steglich conditions the more efficient method. Gratifyingly, the SnCl2*2H2O promoted azide reduction/ O→N acyl migration worked well for azido esters where R ≠ H, and amido alcohols ((±)-87b-e) were synthesized in yields of 70-84% (Table 14). The synthetic route devised also performed well on a gram scale.

O R OH O O O OH O a b O O O N N HN 3 3 R (±)-81 (±)-86 (±)-87 O

Table 14. Synthesis of the amido alcohols 87b-e. Conditions: a) RCH2COOH, DCC,

DMAP, DCM, rt, 1 h; b) i) SnCl2*2H2O, rt, 1 h, ii) NaHCO3, MeOH:H2O or 1,4- dioxane:H2O, rt, 18 h. Entry R Compound (% yield) Compound (% yield) 1 H 86a (96)a 87a (-) 2 cPr 86b (95) 87b (71)b 3 Ph 86c (95) 87c (70)b b 4 mCF3Ph 86d (90) 87d (80) 5 2-thienyl 86e (91) 87e (84)c aAc2O/DMAP was used, bUsing 1,4-dioxane:H2O instead of MeOH:H2O gave lower yield, c1,4-dioxane:H2O was used, MeOH:H2O only gave 50% yield

The initial attempt to prepare the corresponding thiol amide of (±)-87c with 0.6 eq of Lawesson’s reagent afforded the ring closed (±)-88b as the sole product in 80% yield (Table 15, entry 2). The amido alcohols (±)-87a,c, and (±)-87d were subsequently directly converted to the desired 5-substituted Δ2-thiazolines (±)-88a-d under these conditions in yields of 71-80% (Table 15).

OH O R a S S S O O P P O HN N S S R Lawesson's reagent (±)-87 O O (±)-88 O Table 15. Ring closure of the amido alcohols to the Δ2-thiazolines with Lawesson’s reagent. Conditions: a) 0.6 eq Lawesson’s reagent, toluene, reflux, 1 h. Entry Compound R Yield (%) 1 88a cPr 71 2 88b Ph 80

3 88c mCF3Ph 72 4 88d 2-thienyl 74

To further investigate the scope and limitation of this route, a series of compounds in which the phenyl substituent was exchanged for various aromatic moieties was made. The α,β-unsaturated esters were not commercially available but were easily synthesized via the Horner— Wadsworth—Emmons reaction in excellent yields and trans selectivities (Table 16). Following the same route as for the methyl cinnamate worked well for all examples, and the desired azido alcohols (±)92a-d were obtained in 65-82% yields (Table 16).

O a O b OH O c OH O R H R O R O R O OH ONs (±)-89 (±)-90 (±)-91 OH O d R O

N3 (±)-92 Table 16. Synthesis of differently substituted azido alcohols. Conditions: a)

Trimethyl phosphonoacetate, NaH, THF, rt; b) K2OsO4*2H2O, NMO,

MeCN:acetone:H2O 1:1:1, rt; c) NsCl, TEA, 0 °C; d) NaN3, DMF, 40 °C. Entry R Compound Compound Compound Compound (% yield) (% yield) (% yield) (% yield) 1 pFPh 89a (98) 90a (65) 91a (63) 92a (65) 2 mMeOPh 89b (96) 90b (75) 91b (70) 92b (82) 3 Bn 89c (62)a 90c (80) 91c (62) 92c (79) 4 2-thienyl 89d (98) 90d (73) 91d (54) 92d (78) aFreshly distilled aldehyde was required for the reaction to work.

The Steglich esterification and the SnCl2*2H2O promoted azide reduction/ O→N acyl migration proceeded well for azido alcohols (±)92a-d (Table 17), while the following ring-closure to the 5-substituted Δ2-thiazolines with

Lawesson’s reagent was troublesome for the benzyl substituted compound (±)-95c that was obtained in only 24% yield (Table 17, entry 3). However the reaction proceeded smoothly with the other aryl/heteroaryl substituted amido alcohols, with the corresponding 5-substituted Δ2-thiazolines (±)- 95a,b,d isolated with yields of 71%, 80% and 65% respectively (Table 17, entries 1,2 and 4).

OH O O O OH O a b c S R R O R O R O N N N HN 3 3 O (±)-92 (±)-93 O (±)-94 O (±)-95 Table 17. Synthesis of different 5-substituted Δ2-thiazolines. Conditions: a)

Cyclopropylacetic acid, DCC, DMAP, DCM, rt, 1 h; b) i) SnCl2*2H2O, rt, 1 h, ii)

NaHCO3, MeOH:H2O or 1,4-dioxane:H2O, rt, 18h; c) 0.6 eq Lawesson’s reagent, toluene, reflux, 1 h. Entry R Compound Compound Compound (% yield) (% yield) (% yield) 1 pFPh 93a (85) 94a (83) 95a (71) 2 mMeOPh 93b (94) 94b (80) 95b (80) 3 Bn 93c (75) 94c (77) 95c (24) 4 2-thienyl 93d (81) 94d (78) 95d (65)

To prepare target 5-phenyl Δ2-thiazolines asymmetrically via this route, the Sharpless asymmetric dihydroxylation with the commercially available AD-α

/AD-β mixes in tBuOH:H2O (1:1) was employed for the initial dihydroxylation. Further conversion of the dihydroxy compounds (+)-79 and (-)-79 to the nosylates (+)-80 and (-)-80, followed by NaN3 displacement afforded the azido alcohols (+)-81 and (-)-81 in 73% and 70% yields respectively and excellent enantiomeric excesses of 99% (Scheme 20).

OH O OH O OH O c d O O O

a OH ONs N3 (-)-79 83% (-)-80 76% (+)-81 70% O ee 99% ee 99% ee 99%

OH O OH O OH O b c d O O O N OH ONs 3 (+)-80 79% (-)-81 73% (+)-79 85% ee 99% ee 99% ee 99% Scheme 20. Asymmetric synthesis of the azido alcohols. Conditions a) AD-mix-β,

MeSO2NH2, tBuOH:H2O 1:1, rt; b) AD-mix-α, MeSO2NH2, tBuOH:H2O 1:1, rt; c)

NsCl, TEA, DCM, o °C; d) NaN3, DMF 40 °C.

Azido alcohols (+)-81 and (-)-81 were esterified under Steglich conditions, followed by the SnCl2*2H2O induced azide reduction/O→N acyl migration reaction to generate the amido alcohols (+)-87b,e and (+)-87b,e in good yields and excellent ee of 99% (Table 18). The subsequent ring closure with 0.6 eq of Lawesson’s reagent proceeded in yields of 71-80% (Table 18, entries 9-12), but some epimerization was observed in this step (e.g. (-)-87b, 99% ee → (-)-88a, 80% ee), with the 5-substituted Δ2-thiazolines obtained in 71- 88% ee (Table 18, entries 9-12).

O R R OH O O O OH O S a b c O O O N N3 N3 HN R O (+)-81 O O (-)-86b R = cPr (-)-86e R = 2-thienyl (-)-87b R = cPr (-)-88a R = cPr (-)-87e R = 2-thienyl (-)-88d R = 2-thienyl O R R OH O O O OH O S a b c O O O N N3 N3 HN R O (-)-81 (+)-86b R = cPr O O (+)-86e R = 2-thienyl (+)-87b R = cPr (+)-88a R = cPr (+)-87e R = 2-thienyl (+)-88d R = 2-thienyl Table 18. Asymmetric synthesis of 5-phenyl substituted Δ2-thiazolines (-)-88a,d and (+)-88a,d. Conditions: a) RCH2COOH, DCC, DMAP, DCM, rt, 1 h; b) i)

SnCl2*2H2O, rt, 1 h, ii) NaHCO3, MeOH:H2O or 1,4-dioxane:H2O, rt, 18 h; c) 0.6 eq Lawesson’s reagent, toluene, reflux, 1 h. Entry Compound Yield (%) ee (%)a 1 (-)-86b 92 99 2 (-)-86e 91 99 3 (+)-86b 93 99 4 (+)-86e 94 99 5 (-)-87b 72 99 6 (-)-87e 82 99 7 (+)-87b 73 99 8 (+)-87e 80 99 9 (-)-88a 74 80 10 (-)-88d 71 88 11 (+)-88a 80 71 12 (+)-88d 72 83 aDetermined by chiral chromatography

The enantiomerically enriched 5-substituted Δ2-thiazolines (-)-88a,d and (+)-88a,d were utilized in the acid induced acylketene/imine cyclocondensation with acyl Meldrum’s acid 96 to afford the 2-phenyl ring- fused 2-pyridones (-)-97a,b and (+)-97a,b in ee of 70-88% (Table 19).

R R S a S N N O O O O O (-)-88a R = cPr (-)-97a R = cPr HO (-)-88d R = 2-thienyl (-)-97b R = 2-thienyl O O 96 R R O O S S a N N

O O O O O (+)-88a R = cPr (+)-97a R = cPr (+)-88d R = 2-thienyl (+)-97b R = 2-thienyl Table 19. Acid induced ketene/imine cyclocondensation of the asymmetric Δ2- thiazolines, conditions a) 91, TFA, MWI 140 °C for 2 min, DCE Entry Compound Yield (%) ee (%) 1 (-)-97a 81 77 2 (-)-97b 83 88 3 (+)-97a 80 70 4 (+)-97b 85 82

As can be seen from Tables 18 and 19, epimerization occured in the ring closing reaction with Lawesson’s reagent. Consequently a set of reactions in which the amount of Lawesson’s reagent was varied was performed; the amido alcohol (-)-87b was selected as a model substance (Table 20). Lower amounts of Lawesson’s reagent gave lower yield and ee (Table 20, entry 1). However, using 1 eq of Lawesson’s reagent improved both the yield and the ee significantly (Table 20, entry 4). To the best of our knowledge there are no previous reports on direct ring closures of amido alcohols yielding this type of 2,4,5-trisubstituted Δ2-thiazolines with high enantiomeric purity.

OH O Lawesson's reagent S O N HN Toluene, reflux 1 h O O O (-)-87b (-)-88a Table 20. Results of varying amount of Lawesson’s reagent Entry LR (eq) Temp. Yield (%) ee (%)a 1 0.5 refluxb 50 65 2 0.6 rt-refluxc 75 73 3 0.6 refluxb 78 75 4 1.0 refluxb 90 97 aDetermined by chiral chromatography on a Whelk-O1 column, bThe oilbath was preheated to 120 °C before the reaction was started, cThe reaction was heated to reflux starting fron rt, and refluxing continued for 1 h.

These observations supports the mechanism published by Nishio.95b The conversion of alcohols to thiols with Lawesson’s reagent is known to proceed with retention of configuration.97 The thiol amide formed can either undergo a second thiolation of the amide with a second equivalent of Lawesson’s reagent (i.e A, Scheme 21) and subsequent cyclization to form the enantiomerically pure Δ2-thiazoline, or a syn-elimination facilitated by compound B to generate the α,β-unsaturated ester (Scheme 21). This α,β- unsaturated ester can potentially undergo a conjugate addition with a sulfur nucleophile (i.e C, Scheme 21) to generate the racemic thiol amide (Scheme 21). Thiol additions to Michael acceptors are known to precede with high diastereospecificity even at increased temperatures (i.e. 110 °C).98 The diasteriomeric control in this reaction is explained by the selective protonation from the opposite side of the sulphur substituent in the transition state enolate (shown in inlay Scheme 21) due to stereoelectronic effects.98 Alternatively, the α,β-unsaturated ester might undergo thiolation of the amide and a subsequent 5-endo-trig cyclization to give the racemic Δ2- thiazoline. Such 5-endo-trig cyclizations are known to be disfavoured according to “Baldwin’s rules” for first row element based nucleophiles (i.e. oxygen and nitrogen). However, such ring closures with the larger second row elements (i.e. sulfur) are viable.99

S B P O Ar S OH O SH O P S O O HN HN O A R S R C O P O HO Ar Enantiomerically HS O pure A O O O B HN HN A R B R B S O C Conjugate B SH O addition R O SH O HN S R O N S HN R Enantiomerically O O Racemic O pure Racemic

-H2S R HO B R S S HN N S Racemic O P O O HO Ar O HO

SH SH O R O +H+ HN O O HN Ph H R O

Scheme 19. Plausible mechanism for Δ2-thiazoline formation from amido alcohols with Lawesson’s reagent.

In conclusion, robust and scalable synthetic pathways to the highly enantiomerically enriched 2,4,5-trisubstituted Δ2-thiazolines have been developed. These compounds were made via a Sharpless asymmetric dihydroxylation, and an SnCl2*2H2O induced azide reduction/O→N acyl migration reaction as key steps. The ring closure of the amido alcohols was shown to work well with 0.6 eq of Lawesson’s reagent, but was found to give some epimerization. However using 1 eq of Lawesson’s reagent ameliorated this and the Δ2-thiazolines can be obtained in excellent yields and stereochemical purity (exemplified by compound (-)-88a Table 20, entry 4). The synthetic route performed well for 5-aryl/heteroaryl substituted Δ2- thiazolines and the obtained products were further reacted in an acylketene/imine cyclocondensation reaction to yield enantiomerically enriched 2-substituted thiazolino ring-fused 2-pyridones. Moreover, this synthetic route provides facile access to unnatural cysteine and serine analogues, as well as opening up possibilities to construct 5-substituted Δ2- thiazoline containing chiral ligands and analogues of Δ2-thiazoline containing natural products.

2-Furanone or 2-pyrone ring-fused tricyclic scaffolds (Paper VII)

In parallel with our research into finding new and more efficient ways to substitute the 2-pyridone core, we are also interested in central fragment manipulations to create new types of peptidomimetic scaffolds.38, 79, 100 In this paper we present synthetic routes to new 2-furanone or 2-pyrone containing tricyclic peptidomimetic scaffolds. These scaffolds were made via a selective intramolecular acid induced Pd-catalyzed 5-exo-dig or 6-endo-dig cyclization of a carboxylic acid onto an alkyne. 2-Furanones and 2-pyrones have previously been synthesized via 5-exo-dig or 6-endo-dig cyclizations of carboxylic acids/esters onto acetylenes.101 We envivioned a synthesis of the desired acetylene substituted compounds from a Sonogashira coupling of the known brominated compounds 6137 followed by an acid induced cyclization between the methyl ester and the alkyne (Scheme 22).

Sonogashira Sonogashira coupling coupling R1 R1 R1

R S R S R S Br N N N O O O O O O O O O Acid induced 1 5-exo-dig 61a R = 1-Naphthyl, R = cPr Acid induced 1 cyclization 61b R = 1-Naphthyl, R = mCF3Ph 6-endo-dig cyclization Scheme 22. Retrosynthetic analysis to the ring-fused tricyclic compounds

The Sonogashira coupling performed well with 61a, Pd(PPh3)2Cl2, CuI, TEA and TMSA in DMF under microwave irradiation, followed by a TMS deprotection with K2CO3 in MeOH:THF (4:1) to give the desired acetylene functionalized methyl ester 66 in 73% yield. All attempts of the following ring-closure to access 98 and/or 99 failed. Only starting material was obtained even when TFA was used as the reaction solvent (Scheme 23).

S a S Br N N

O O O O O O 61a 66

S S N N O O O O O O 98 99

Scheme 23. Alkyne functionalization and attempted ring closure. Conditions: a) i)

Pd(PPh3)2Cl2, TMSA, CuI, TEA, DMF, MWI 110 oC for 10 min; ii) K2CO3, MeOH:THF 4:1, rt, 15 min, 73%.

Our anticipation was that the corresponding carboxylic acid might ring-close more easily, hence an acid labile ester was desirable to prevent an extra hydrolysis step, particularly as this conversion had previously proved difficult with α,β-unsaturated esters.60 We anticipated that the TMSE ester would be a good choice and stable enough to withstand the conditions used throughout the synthetic sequence. The syntheses of the TMSE ester protected intermediates 101a,b from pyridones 74 and 100 were straightforward (Scheme 24): hydrolysis and conversion to the corresponding acid chloride with oxalyl chloride and catalytic amounts of DMF, followed by quenching by TMSE-OH afforded the racemic TMSE esters 101a,b in excellent yields. The oxidation/bromination reaction previously used for methyl esters60 performed well with the TMSE esters once MeOH was exchanged for TMSE-OH to prevent transesterification. TMSE esters have been reported to be labile in the presence of NaH,102 however no such reaction was observed in these examples. The Sonogashira coupling with TMSA and subsequent TMS deprotection worked smoothly and the desired TMSE protected acetylene activated intermediates 103a,b were isolated in yields of 61% and 62% respectively (Scheme 24).

R1 R1 R1 S a S b S Br N N N

O O O O O O O O O 1 100 R = cPr 1 1 1 101a R = cPr 102a R = cPr 74 R = mCF3Ph 1 Si 1 Si 101b R = mCF3Ph 102b R = mCF3Ph R1 c S N

O O O 103a R1 = cPr 103b R1 = mCF Ph Si 3 Scheme 24. Synthesis of the TMSE protected alkyne functionalized comounds.

Conditions: a) i) 1 M LiOH (aq), THF, rt, 20 min, ii) (COCl)2, cat. DMF, TMSE-OH,

DCM, rt, 30 min; b) NaH, BrCCl3, TMSE-OH, MeCN, rt, 10 h; c) i) Pd(PPh3)2Cl2,

TMSA, CuI, TEA, DMF, MWI 110 oC for 10 min, ii) K2CO3, MeOH:THF 4:1.

Cyclizations of carboxylic acids/esters onto alkynes are known to proceed with bad selectivity and can demand high concentrations of acids in certain cases.101 While the cyclization of compound 103a proceeded smoothly with 20% TFA in DCM at rt, a 1:1 mixture of the 5-exo-dig (98) and 6-endo-dig (99) cyclization products was obtained. We therefore undertook a catalyst screen to gain selectivity in this reaction (Table 21).

S a S S

N N + N O O O O O O O O O 103a 98 99 Si

Table 21. Catalyst screen for the ring closing reaction. Conditions: a) DCM:TFA 85:15, 5 mol% catalyst, 5 mol% ligand, rt. The reactions were performed on a 10 mg/ml scale

Entry Catalyst Ligand Time Ratio Conversion (h) 98:99 (isolated yield) (%) a 1 Pd(OAc)2 2 80:20 100 b a 2 Pd(OAc)2 DPPF 2 95:5 100 c 3 Pd(OAc)2 DPPP 1 >99% 98 100 (88) 4 Pd-IPr-NHCd 2.5 10:90 100 (88) 5 Pd-SIPr-NHCe 2.5 5:95 100a a 6 PdCl2 2.5 50:50 80 7 Pd(PPh3)2Cl2 2.5 Complex mixture

8 Pd(PPh3)4 2 Complex mixture f a 9 Pd2(dba)3*CHCl3 1.5 80:20 100 a 10 PPh3AuCl/AgSBF6 3 95:5 100 a 11 AuCl3 3 50:50 100 aNo byproducts detected by LC-MS, bDPPF = 1,1’-bis(diphenylphosphino)ferrocene, cDPPP = 1,3-bis(diphenylphosphino)propane, dPd-IPr-NHC = [1,3-Bis(2,6- Diisopropylphenyl)imidazol-2-ylidene](3-chloropyridyl)palladium(II) dichloride, ePd-SIPr-NHC = [1,3-Bis(2,6-diisopropylphenyl)imidazolidene](3-chloropyridyl) palladium(II) dichloride, fdba = dibenzylideneacetone

As Table 21 reveals most of the catalysts tested induced the 5-exo-dig cyclization (98), with the Pd(OAc)2/DPPP catalytic system the most efficient (Table 21, entry 3). A reaction with the Pd(OAc)2/DPPP catalytic system in DCM:TFA 95:5 was also performed, but these conditions gave less selectivity with about 5% of the 6-endo-dig product 99 was formed. Switching to NHC- based Pd-catalysts gave up to 95% selevtivity in preference for the 6-endo- dig product 99 (Table 21, entry 4-5). These results encouraged us to investigate Lewis acid additives in the reaction, and a set of tetra-, tri- and divalent Lewis acids were screened together with Pd-IPr-NHC (Table 22).

S a S S

N N + N O O O O O O O O O 103a 98 99 Si

Table 22. Lewis acids screen of the ring closing reaction, conditions a) 5 mol% Pd- IPr-NHC, Lewis acid, DCM, rt, the reactions were performed on a 10 mg/ml scale.

Entry Lewis acid (10 eq) Ratio 98:99 Time (h) Comment

1 ZrCl4 18 Complex mixture a 2 TiCl4 18 Only ester deprotection a 3 SnCl4 18 Complex mixture

4 TiCl3 18 Only ester deprotection

5 InCl3 20:80 18 70% starting material

6 SbF3 18 No reaction a 7 BF3*Et2O 100% 99 3 100% conversion

8 SnCl2 18 No reaction aDistilled before use

In this screen, BF3*Et2O was the only Lewis acid which proved efficient (Table 22, entry 7). When the reaction was also performed without Pd- catalyst, under these conditions only deprotection of the TMSE ester was observed. No cyclization occurred even when the amount of BF3*Et2O was increased to 20 mol%. Exchanging the cPr substituent in the 8-position for a mCF3Ph substituent of the ring-fused 2-pyridone (103b) had an unexpectedly large impact on the reaction rate, and only 20% conversion to the 6-endo-dig:5-exo-dig cyclized products 104:105 (1:1) was observed after 18 h reaction at rt in DCM:TFA (80:20) without any catalyst present. On the other hand in the presence of a Pd-catalyst, the reaction proceeded with excellent selectivities for the 6-endo-dig (104) or 5-exo-dig (105) cyclizations in 3 and 1 h respectively (Table 25).

F F F F F F F F F

S a S b S N N N O O O O O O O O O 105 104 103b Si

Scheme 19. Ring closing reaction with the electron deficient mCF3Ph substituent.

Conditions: a) Pd-IPr-NHC, DCM:BF3*Et2O 95:5, rt, 3 h, 91%; b) Pd(OAc)2, DPPP, DCM:TFA 85:15, rt, 1 h, 85%.

The mechanism of the Pd-IPr-NHC/DCM:BF3*Et2O induced cyclization is still unknown, wheras the Pd(OAc)2/DPPP catalyzed reaction in DCM:TFA might proceed via the Pd(II) catalytic cycle previously published for similar systems by Fujiwara and coworkers (Scheme 26).103

1 1 R R A S TFA S N N

O OTMSE O OH O O

Ph Ph Ph Ph R1 P P R1 Ph Pd Ph TFAO P S (OTFA)2 S Pd -OTFA -H+ P N N +OTFA Ph Ph O OH O O O HO

R1 R1 Ph Ph Ph Ph S TFAO P S P P Pd Ph Pd Ph N N TFAO OTFA O P O O Ph Ph TFA A O A O O Scheme 26. Plausible mechanism for the Pd(OAc)2/DPPP catalyzed 5-exo-dig cyclization

Considering the great selectivities and conversions in the synthesis of the 5- exo-dig (98 and 105) and 6-endo-dig (99 and 104) compounds, we were encouraged to investigate the reaction on internal acetylenes. Consequently a

set of internal acetylenes (106a-f) bearing substituents with different electronic properties was synthesized via the Sonogashira coupling (Table 23).

S a S Br R N N

O O O O O O 102a 106 Si Si

Table 23. Alkyne functionalization of the 2-position via the Sonogashira coupling.

Conditions: a) Pd(PPh3)2Cl2, alkyne, CuI, TEA, DMF, MWI 110 °C, 10 min.

Entry R Yield (%) Compound 1 nPr 86 106a 2 cPr 84 106b 3 Ph 95 106c 4 pMeOPh 89 106d

5 mCF3Ph 86 106e 6 3-thienyl 95 106f

The internal acetylenes were investigated in the cyclization reaction with both catalytic systems (i.e. Pd(OAc)2/DPPP and Pd-IPr-NHC) as well as without any catalyst. All the conditions tested produced the 6-endo-dig cyclization product 107 in excellent yields (Table 24).

S a S R N N R O O O O O O 106 107 Si

Table 24. Ring closure of internal acetylenes. Conditions: a) A: DCM:TFA (80:20), or B: 5 mol% Pd(OAc)2, 5 mol% DPPP, DCM:TFA (85:15), or C: 5 mol% Pd-IPr-NHC,

DCM:BF3*Et2O (95:5).

Entry Compound R Conditions Time (h) Conversion (isolated yield) (%) 1 107a nPr A 28 100 (99) 2 107a nPr B 1 100a 3 107a nPr C 2 100a 4 107b cPr A 1 100 (99) 5 107b cPr B 1 100a 6 107b cPr C 4 100a 7 107c Ph A 18 100 (98) 8 107c Ph B 1 100a 9 107c Ph C 18 100a 10 107d pMeOPh A 1 100 (99) 11 107d pMeOPh B 1 100a 12 107d pMeOPh C 2.5 100a

13 107e mCF3Ph A 96 90 (88) a 14 107e mCF3Ph B 2 100 a 15 107e mCF3Ph C 2 100 16 107f 3-thienyl A 20 100 (99) 17 107f 3-thienyl B 1 100a 18 107f 3-thienyl C 1.5 100a aNo byproduct detected by LC-MS

The acetylene with the electron poor mCF3Ph substituent did not reach full conversion even after 96 h reaction time at rt (Table 24, entry 13). In contrast, the electron rich pMeOPh substituent (Table 24, entry 10) and the cPr substituent (Table 24, entry 4) achieved full conversion after 1 h, which lead us to believe that this reaction occurs via a cationic intermediate. The great reaction rate enhancement observed by the cPr substituted acetylene under none catalysed conditions (Table 24, entry 4) compared to it’s straight chain counterpart (i.e. nPr, Table 24, entry 1) can be attributed to it’s “sp2- like” character.104

In conclusion, synthetic pathways to the TMSE protected esters 103a-b and 106a-f have been developed which were successfully used in an acid induced 5-exo-dig or 6-endo-dig cyclization onto an alkyne to yield 2-furanone or 2- pyrone ring fused tricyclic systems in excellent regioselectivities. The

Pd(OAc)2/DPPP catalytic system in DCM:TFA gave preference for the 5-exo- dig cyclization (98), whereas the Pd-IPr-NHC based catalytic system in

DCM:BF3*Et2O gave preference for the 6-endo-dig cyclization (99). The cyclization reaction worked well for both terminal and internal alkynes but in the latter case only the 6-endo-dig products (107a-f) were isolated, regardless of the catalytic system employed. This reaction proved effective for aromatic, heteroaromatic, cycloaliphatic and aliphatic substituents on the acetylene.

Concluding remarks

The resistance to antibiotics displayed by an ever growing number of strains of microbes in the modern world is an imminent threat to public health. The search for new types of antibiotics to treat bacterial infections accordingly becomes more and more urgent every day. Research in this area must go on both in terms of finding replacements for old antibiotics where resistance is widespread, as well as finding novel compounds which strike unutilised targets. As a contribution to solving that puzzle, the class of ring-fused 2- pyridones was presented herein. This class of compound is known to inhibit the chaperone usher pathway, responsible for assembly of pili in UPEC. To explore and fine-tune the biological efficacy of these compounds more effectively, in addition to increasing our knowledge of their mode of action, fast and efficient synthetic pathways are necessary. As a part of that research we have developed scaffolds that allow late stage diversification in positions 6, 8 (Paper I) and 7 (Paper II). The tool compounds prepared have been successfully applied in various applications in our research. Firstly, these scaffolds have been used for the synthesis of fluorescent compounds to further study the complex mechanisms of bacterial virulence (Paper III). Secondly, the developed bromo-iodo scaffold allowed introduction of triazole functionalities in the 8-position (Paper IV), and have contributed to the discovery of the first reported accelerators of CsgA polymerization into amyloid fibres (Paper V). It is worth emphasizing that the accelerators would most probably not have been discovered without this key bromo-iodo scaffold. Thirdly, the type of halo-methyl scaffolds developed in Paper II have been used not just for diversification of position 7 as presented in Papers II and III, but have also been used as intermediate in manipulations of the central fragment. Lastly both scaffolds have been used to attach biotin containing linkers to different positions of the ring-fused 2-pyridones in an ongoing project to more precisely identify the target proteins involved in the observed biological activities. Furthermore, synthetic pathways to reach enantiomerically enriched key compounds have been developed via the construction of asymmetric 2,4,5-trisubstituted Δ2-thiazolines (Paper VI). Prior to this work, reports on this type of Δ2-thiazolines in the literature were scarce; this framework possesses interesting properties for future use as chiral ligands for asymmetric catalysis as well as in Δ2-thiazoline based natural product analogues. Methods to produce either 2-furanone or 2- pyrone ring-fused tricyclic systems from the same starting material in excellent yields and selectivities were also developed (Paper VII). Despite the lack of a free carboxylic acid in these compounds they still exhibit interesting properties as starting materials for further transformations, or as rigidified peptidomimetics for future applications in chemical biology.

Acknowledgements

Jag känner mig priviligerad som har haft möjliheten att få samarbeta med så många duktiga och trevliga människor under min doktorandtid. Här är några jag vill rikta ett speciellt tack till.

Min handledare Fredrik Almqvist för att du anställde mig som doktorand och för att du har uppmuntrat till arbete enligt modellen ”frihet under ansvar”. Och sist men inte minst för att du är snäll, ödmjuk och lätt att jobba för.

Mikael E, Anna och Dan för att ni har tagit er tid att deltaga vid mina årliga uppföljningssamtal. Tack till alla mina trevliga kollegor på AstraZeneca R&D Mölndal för att ni inspirerat mig ännu mer och gjort ämnet organisk syntes ännu roligare. Alla mina labkollegor: Magnus, Erik C, Sofie, Syam, James, Tomas, Dang, Munawar, Deepak, Pardeep, Ida, Hasse, Sajal, Thomas och Usma samt mina projekt- och ex-jobbare: Karl, Anders, Maximillian, Jasmine, Jun och Lianpao. Och givetvis alla andra på avdelningen/institutionen som gjort min doktorandtid till en minnesvärd tid. James for proofreading of my thesis, Hanna U och Emma för ett fantastiskt snabbt och bra arbete med biologisk testning av mina föreningar i pili och curli projekten. Våra samarbetspartners Scott och Jerry i Saint Louis samt Matt och hans grupp i Michigan. Erik R och Lennart för fluorescence mätningar, Mattias för hjälp med NMR instrumentet när det krånglat, Mackan och Weixing för hjälp med den preparativa HPLC:n när den krånglat eller efter att jag har krökt nålen, Sergey för hjälp med HRMS instrumentet och Hanna N för hjälp med kirala separationer. Apotekarsocieteten, Helge Ax:son Johnsons stiftelse och Ångpanneföreningens forskningsstiftelse för att ni gjort det ekonomiskt möjligt för mig att deltaga vid ACS mötena i San Fransisco, Denver, Philadelphia och New Orleans under min doktorandtid. Tack till Tomas, Neas, Mattias, Karl, John, Mikael H, Markus och Sara som har varit stammisar vid ”fredagsölen” och grillkvällar där det mesta mellan himmel och jord har diskuterats. Tack till David och Jonas för att ha varit ständiga lunch partners. Och sist men inte minst min familj Märit, Håkan, Pierre och Rosita, mina syskonbarn Joel och Linnea, Frida och Erik och erat kommande tillskott till familjen, min mormor Evy som med stor sorg lämnade oss 2010. Mina vänner Christopher och Caroline samt Markus och Danielle som alltid ställer upp och finns till hands.

References

1. Wöhler, F., Ueber künstliche Bildung des Harnstoffs. Annalen der Physik 1828, 87 (2), 253-256.

2. Kolbe, H., Untersuchungen über die Elektrolyse organischer Verbindungen. Justus Liebigs Annalen der Chemie 1849, 69 (3), 257-294.

3. (a) Baeyer, A.; Villiger, V., Einwirkung des Caro'schen Reagens auf Ketone. Berichte der deutschen chemischen Gesellschaft 1899, 32 (3), 3625-3633; (b) Baeyer, A.; Villiger, V., Ueber die Einwirkung des Caro'schen Reagens auf Ketone. Berichte der deutschen chemischen Gesellschaft 1900, 33 (1), 858-864.

4. Grignard, V., Sur quelques nouvelles combinaisons organométaliques du magnésium et leur application à des synthèses d'alcools et d'hydrocabures. Comptes rendus hebdomadaires des séances de l'Académie des Sciences 1900, 130, 1322-1325.

5. Diels, O.; Alder, K., Synthesen in der hydroaromatischen Reihe. Justus Liebigs Annalen der Chemie 1928, 460 (1), 98-122.

6. (a) Kagan, H. B.; Riant, O., Catalytic asymmetric Diels Alder reactions. Chemical Reviews 1992, 92 (5), 1007-1019; (b) Takao, K.-i.; Munakata, R.; Tadano, K.-i., Recent Advances in Natural Product Synthesis by Using Intramolecular Diels−Alder Reactions. Chemical Reviews 2005, 105 (12), 4779-4807.

7. Woodward, R. B.; Cava, M. P.; Ollis, W. D.; Hunger, A.; Daeniker, H. U.; Schenker, K., The total synthesis of strychnine. Journal of the American Chemical Society 1954, 76 (18), 4749-4751.

8. (a) Wittig, G.; Haag, W., Über Triphenyl- phosphinmethylene als olefinbildende Reagenzien Chemische Berichte 1955, 88 (11), 1654-1666; (b) Wittig, G.; Schöllkopf, U., Über Triphenyl-phosphin-methylene als olefinbildende Reagenzien. Chemische Berichte 1954, 87 (9), 1318-1330.

9. (a) Brown, H. C.; Zweifel, G., Bis-3-methyl-2- butylborane as a selective reagent for steric control of the direction of hydroboration. Journal of the American Chemical Society 1960, 82 (12), 3222-3223; (b) Brown, H. C.; Zweifel, G., Hydroboration as a

convenient procedure for the asymmetric synthesis of alcohols of high optical purity. Journal of the American Chemical Society 1961, 83 (2), 486-487.

10. Olah, G.; Kuhn, S.; Olah, J., 416. Aromatic substitution. Part III. Alkylation of aromatic compounds by the boron trifluoride- catalysed reaction of alkyl fluorides. Journal of the Chemical Society (Resumed) 1957, 2174-2176.

11. Merrifield, R. B., Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide. Journal of the American Chemical Society 1963, 85 (14), 2149-2154.

12. (a) Kochi, J. K.; Tamura, M., Mechanism of the silver- catalyzed reaction of Grignard reagents with alkyl halides. Journal of the American Chemical Society 1971, 93 (6), 1483-1485; (b) Kochi, J. K.; Tamura, M., Alkylcopper(I) in the coupling of Grignard reagents with alkyl halides. Journal of the American Chemical Society 1971, 93 (6), 1485-1487; (c) Tamura, M.; Kochi, J. K., Vinylation of Grignard reagents. Catalysis by iron. Journal of the American Chemical Society 1971, 93 (6), 1487-1489.

13. Mizoroki, T.; Mori, K.; Ozaki, A., Arylation of Olefin with Aryl Iodide Catalyzed by Palladium. Bulletin of the Chemical Society of Japan 1971, 44 (2), 581-581.

14. Heck, R. F.; Nolley, J. P., Palladium-catalyzed vinylic hydrogen substitution reactions with aryl, benzyl, and styryl halides. The Journal of Organic Chemistry 1972, 37 (14), 2320-2322.

15. Corriu, R. J. P.; Masse, J. P., Activation of Grignard reagents by transition-metal complexes. A new and simple synthesis of trans-stilbenes and polyphenyls. Journal of the Chemical Society, Chemical Communications 1972, (3), 144a-144a.

16. Tamao, K.; Sumitani, K.; Kumada, M., Selective carbon- carbon bond formation by cross-coupling of Grignard reagents with organic halides. Catalysis by nickel-phosphine complexes. Journal of the American Chemical Society 1972, 94 (12), 4374-4376.

17. Sonogashira, K.; Tohda, Y.; Hagihara, N., A convenient synthesis of acetylenes: catalytic substitutions of acetylenic hydrogen with bromoalkenes, iodoarenes and bromopyridines. Tetrahedron Letters 1975, 16 (50), 4467-4470.

18. King, A. O.; Okukado, N.; Negishi, E.-i., Highly general stereo-, regio-, and chemo-selective synthesis of terminal and internal conjugated enynes by the Pd-catalysed reaction of alkynylzinc reagents with alkenyl halides. Journal of the Chemical Society, Chemical Communications 1977, 0 (19), 683-684.

19. (a) Milstein, D.; Stille, J. K., A general, selective, and facile method for ketone synthesis from acid chlorides and organotin compounds catalyzed by palladium. Journal of the American Chemical Society 1978, 100 (11), 3636-3638; (b) Stille, J. K., The Palladium-Catalyzed Cross-Coupling Reactions of Organotin Reagents with Organic Electrophiles [New Synthetic Methods (58)]. Angewandte Chemie International Edition in English 1986, 25 (6), 508-524.

20. Miyaura, N.; Yamada, K.; Suzuki, A., A new stereospecific cross-coupling by the palladium-catalyzed reaction of 1- alkenylboranes with 1-alkenyl or 1-alkynyl halides. Tetrahedron Letters 1979, 20 (36), 3437-3440.

21. (a) Nicolaou, K. C.; Bulger, P. G.; Sarlah, D., Palladium- Catalyzed Cross-Coupling Reactions in Total Synthesis. Angewandte Chemie International Edition 2005, 44 (29), 4442-4489; (b) Torborg, C.; Beller, M., Recent Applications of Palladium-Catalyzed Coupling Reactions in the Pharmaceutical, Agrochemical, and Fine Chemical Industries. Advanced Synthesis & Catalysis 2009, 351 (18), 3027-3043.

22. (a) Knowles, W. S.; Sabacky, M. J., Catalytic asymmetric hydrogenation employing a soluble, optically active, rhodium complex. Chemical Communications (London) 1968, 0 (22), 1445- 1446; (b) Vineyard, B. D.; Knowles, W. S.; Sabacky, M. J.; Bachman, G. L.; Weinkauff, D. J., Asymmetric hydrogenation. Rhodium chiral bisphosphine catalyst. Journal of the American Chemical Society 1977, 99 (18), 5946-5952.

23. Miyashita, A.; Yasuda, A.; Takaya, H.; Toriumi, K.; Ito, T.; Souchi, T.; Noyori, R., Synthesis of 2,2'-bis(diphenylphosphino)- 1,1'-binaphthyl (BINAP), an atropisomeric chiral bis(triaryl)phosphine, and its use in the rhodium(I)-catalyzed asymmetric hydrogenation of .alpha.-(acylamino)acrylic acids. Journal of the American Chemical Society 1980, 102 (27), 7932- 7934.

24. (a) Jacobsen, E. N.; Marko, I.; Mungall, W. S.; Schroeder, G.; Sharpless, K. B., Asymmetric dihydroxylation via ligand-accelerated catalysis. Journal of the American Chemical Society 1988, 110 (6), 1968-1970; (b) Katsuki, T.; Sharpless, K. B., The first practical method for asymmetric epoxidation. Journal of the American Chemical Society 1980, 102 (18), 5974-5976; (c) Sharpless, K. B.; Akashi, K., Osmium catalyzed vicinal hydroxylation of olefins by tert-butyl hydroperoxide under alkaline conditions. Journal of the American Chemical Society 1976, 98 (7), 1986-1987.

25. (a) Berrisford, D. J.; Bolm, C.; Sharpless, K. B., Ligand- Accelerated Catalysis. Angewandte Chemie International Edition in English 1995, 34 (10), 1059-1070; (b) Kolb, H. C.; VanNieuwenhze, M. S.; Sharpless, K. B., Catalytic Asymmetric Dihydroxylation. Chemical Reviews 1994, 94 (8), 2483-2547; (c) Sharpless, K. B., Searching for New Reactivity (Nobel Lecture). Angewandte Chemie International Edition 2002, 41 (12), 2024-2032.

26. Jean-Louis Hérisson, P.; Chauvin, Y., Catalyse de transformation des oléfines par les complexes du tungstène. II. Télomérisation des oléfines cycliques en présence d'oléfines acycliques. Die Makromolekulare Chemie 1971, 141 (1), 161-176.

27. (a) Grubbs, R. H.; Brunck, T. K., Possible intermediate in the tungsten-catalyzed olefin metathesis reaction. Journal of the American Chemical Society 1972, 94 (7), 2538-2540; (b) Grubbs, R. H.; Burk, P. L.; Carr, D. D., Mechanism of the olefin metathesis reaction. Journal of the American Chemical Society 1975, 97 (11), 3265-3267.

28. Schrock, R. R., Alkylcarbene complex of tantalum by intramolecular .alpha.-hydrogen abstraction. Journal of the American Chemical Society 1974, 96 (21), 6796-6797.

29. (a) Lozano-Vila, A. M.; Monsaert, S.; Bajek, A.; Verpoort, F., Ruthenium-Based Olefin Metathesis Catalysts Derived from Alkynes. Chemical Reviews 2010, 110 (8), 4865-4909; (b) Nicolaou, K. C.; Bulger, P. G.; Sarlah, D., Metathesis Reactions in Total Synthesis. Angewandte Chemie International Edition 2005, 44 (29), 4490-4527; (c) Vougioukalakis, G. C.; Grubbs, R. H., Ruthenium-Based Heterocyclic Carbene-Coordinated Olefin Metathesis Catalysts†. Chemical Reviews 2010, 110 (3), 1746-1787.

30. (a) Mol, J. C., Industrial applications of olefin metathesis. Journal of Molecular Catalysis A: Chemical 2004, 213 (1), 39-45; (b) Nicola, T.; Brenner, M.; Donsbach, K.; Kreye, P., First Scale-Up to Production Scale of a Ring Closing Metathesis Reaction Forming a 15-Membered Macrocycle as a Precursor of an Active Pharmaceutical Ingredient. Organic Process Research & Development 2005, 9 (4), 513-515.

31. (a) Nicolaou, K. C., The Total Synthesis of Brevetoxin B: A Twelve-Year Odyssey in Organic Synthesis. Angewandte Chemie International Edition in English 1996, 35 (6), 588-607; (b) Nicolaou, K. C.; Rutjes, F. P. J. T.; Theodorakis, E. A.; Tiebes, J.; Sato, M.; Untersteller, E., Total Synthesis of Brevetoxin B. 2. Completion. Journal of the American Chemical Society 1995, 117 (3), 1173-1174.

32. Matsuo, G.; Kawamura, K.; Hori, N.; Matsukura, H.; Nakata, T., Total Synthesis of Brevetoxin-B. Journal of the American Chemical Society 2004, 126 (44), 14374-14376.

33. (a) Dragovich, P. S.; Prins, T. J.; Zhou, R.; Brown, E. L.; Maldonado, F. C.; Fuhrman, S. A.; Zalman, L. S.; Tuntland, T.; Lee, C. A.; Patick, A. K.; Matthews, D. A.; Hendrickson, T. F.; Kosa, M. B.; Liu, B.; Batugo, M. R.; Gleeson, J.-P. R.; Sakata, S. K.; Chen, L.; Guzman, M. C.; Meador, J. W.; Ferre, R. A.; Worland, S. T., Structure-Based Design, Synthesis, and Biological Evaluation of Irreversible Human Rhinovirus 3C Protease Inhibitors. 6. Structure−Activity Studies of Orally Bioavailable, 2-Pyridone- Containing Peptidomimetics. Journal of Medicinal Chemistry 2002, 45 (8), 1607-1623; (b) Golec, J. M. C.; Mullican, M. D.; Murcko, M. A.; Wilson, K. P.; Kay, D. P.; Jones, S. D.; Murdoch, R.; Bemis, G. W.; Raybuck, S. A.; Luong, Y.-P.; Livingston, D. J., Structure-based design of non-peptidic pyridone aldehydes as inhibitors of interleukin-1β converting enzyme. Bioorganic & Medicinal Chemistry Letters 1997, 7 (17), 2181-2186; (c) Sanderson, P. E. J.; Dyer, D. L.; Naylor-Olsen, A. M.; Vacca, J. P.; Gardell, S. J.; Dale Lewis, S.; Lucas Jr, B. J.; Lyle, E. A.; Lynch Jr, J. J.; Mulichak, A. M., L-373,890, An achiral, noncovalent, subnanomolar thrombin inhibitor. Bioorganic & Medicinal Chemistry Letters 1997, 7 (12), 1497-1500; (d) Semple, G.; Ashworth, D. M.; Baker, G. R.; Batt, A. R.; Baxter, A. J.; Benzies, D. W. M.; Elliot, L. H.; Evans, D. M.; Franklin, R. J.; Hudson, P.; Jenkins, P. D.; Pitt, G. R.; Rooker, D. P.; Sheppard, A.; Szelke, M.; Yamamoto, S.; Isomura, Y., Pyridone-based peptidomimetic inhibitors of interleukin-1β-converting enzyme (ICE). Bioorganic & Medicinal Chemistry Letters 1997, 7 (10), 1337-1342; (e) Svensson,

A.; Larsson, A.; Emtenäs, H.; Hedenström, M.; Fex, T.; Hultgren, S. J.; Pinkner, J. S.; Almqvist, F.; Kihlberg, J., Design and Evaluation of Pilicides: Potential Novel Antibacterial Agents Directed Against Uropathogenic Escherichia coli. ChemBioChem 2001, 2 (12), 915- 918; (f) Tamura, S. Y.; Edward Semple, J.; Reiner, J. E.; Goldman, E. A.; Brunck, T. K.; Lim-Wilby, M. S.; Carpenter, S. H.; Rote, W. E.; Oldeshulte, G. L.; Richard, B. M.; Nutt, R. F.; Ripka, W. C., Design and synthesis of a novel class of thrombin inhibitors incorporating heterocyclic dipeptide surrogates. Bioorganic & Medicinal Chemistry Letters 1997, 7 (12), 1543-1548; (g) Verissimo, E.; Berry, N.; Gibbons, P.; Cristiano, M. L. S.; Rosenthal, P. J.; Gut, J.; Ward, S. A.; O’Neill, P. M., Design and synthesis of novel 2-pyridone peptidomimetic falcipain 2/3 inhibitors. Bioorganic & Medicinal Chemistry Letters 2008, 18 (14), 4210-4214; (h) Warner, P.; Green, R. C.; Gomes, B.; Strimpler, A. M., Non-peptidic Inhibitors of Human Leukocyte Elastase. 1. The Design and Synthesis of Pyridone-Containing Inhibitors. Journal of Medicinal Chemistry 1994, 37 (19), 3090- 3099; (i) Åberg, V.; Sellstedt, M.; Hedenström, M.; Pinkner, J. S.; Hultgren, S. J.; Almqvist, F., Design, synthesis and evaluation of peptidomimetics based on substituted bicyclic 2-pyridones— Targeting virulence of uropathogenic E. coli. Bioorganic & Medicinal Chemistry 2006, 14 (22), 7563-7581.

34. (a) Emtenäs, H.; Taflin, C.; Almqvist, F., Efficient microwave assisted synthesis of optically active bicyclic 2-pyridinones via Δ2-thiazolines. Mol Divers 2003, 7 (2-4), 165-169; (b) Emtenäs, H.; Åhlin, K.; Pinkner, J. S.; Hultgren, S. J.; Almqvist, F., Design and Parallel Solid-Phase Synthesis of Ring-Fused 2-Pyridinones That Target Pilus Biogenesis in Pathogenic Bacteria. Journal of Combinatorial Chemistry 2002, 4 (6), 630-639.

35. Åberg, V.; Hedenstrom, M.; Pinkner, J. S.; Hultgren, S. J.; Almqvist, F., C-Terminal properties are important for ring-fused 2-pyridones that interfere with the chaperone function in uropathogenic E. coli. Organic & Biomolecular Chemistry 2005, 3 (21), 3886-3892.

36. (a) Pemberton, N.; Pinkner, J. S.; Jones, J. M.; Jakobsson, L.; Hultgren, S. J.; Almqvist, F., Functionalization of bicyclic 2-pyridones targeting pilus biogenesis in uropathogenic Escherichia coli. Tetrahedron Letters 2007, 48 (26), 4543-4546; (b) Pemberton, N.; Åberg, V.; Almstedt, H.; Westermark, A.; Almqvist, F., Microwave-Assisted Synthesis of Highly Substituted

Aminomethylated 2-Pyridones. The Journal of Organic Chemistry 2004, 69 (23), 7830-7835.

37. Chorell, E.; Das, P.; Almqvist, F., Diverse Functionalization of Thiazolo Ring-Fused 2-Pyridones. The Journal of Organic Chemistry 2007, 72 (13), 4917-4924.

38. Sellstedt, M.; Almqvist, F., Synthesis of a Novel Tricyclic Peptidomimetic Scaffold. Organic Letters 2008, 10 (18), 4005-4007.

39. (a) Cegelski, L.; Pinkner, J. S.; Hammer, N. D.; Cusumano, C. K.; Hung, C. S.; Chorell, E.; Åberg, V.; Walker, J. N.; Seed, P. C.; Almqvist, F.; Chapman, M. R.; Hultgren, S. J., Small- molecule inhibitors target Escherichia coli amyloid biogenesis and biofilm formation. Nat Chem Biol 2009, 5 (12), 913-919; (b) Pinkner, J. S.; Remaut, H.; Buelens, F.; Miller, E.; Åberg, V.; Pemberton, N.; Hedenström, M.; Larsson, A.; Seed, P.; Waksman, G.; Hultgren, S. J.; Almqvist, F., Rationally designed small compounds inhibit pilus biogenesis in uropathogenic bacteria. Proceedings of the National Academy of Sciences 2006, 103 (47), 17897-17902.

40. (a) Boucher, H. W.; Talbot, G. H.; Bradley, J. S.; Edwards, J. E.; Gilbert, D.; Rice, L. B.; Scheld, M.; Spellberg, B.; Bartlett, J., Bad Bugs, No Drugs: No ESKAPE! An Update from the Infectious Diseases Society of America. Clinical Infectious Diseases 2009, 48 (1), 1-12; (b) Clatworthy, A. E.; Pierson, E.; Hung, D. T., Targeting virulence: a new paradigm for antimicrobial therapy. Nat Chem Biol 2007, 3 (9), 541-548.

41. (a) Dodson, K. W.; Pinkner, J. S.; Rose, T.; Magnusson, G.; Hultgren, S. J.; Waksman, G., Structural Basis of the Interaction of the Pyelonephritic E. coli Adhesin to Its Human Kidney Receptor. Cell 2001, 105 (6), 733-743; (b) Mulvey, M. A.; Lopez-Boado, Y. S.; Wilson, C. L.; Roth, R.; Parks, W. C.; Heuser, J.; Hultgren, S. J., Induction and Evasion of Host Defenses by Type 1-Piliated Uropathogenic Escherichia coli. Science 1998, 282 (5393), 1494- 1497; (c) Roberts, J. A.; Marklund, B. I.; Ilver, D.; Haslam, D.; Kaack, M. B.; Baskin, G.; Louis, M.; Möllby, R.; Winberg, J.; Normark, S., The Gal(alpha 1-4)Gal-specific tip adhesin of Escherichia coli P- fimbriae is needed for pyelonephritis to occur in the normal urinary tract. Proceedings of the National Academy of Sciences 1994, 91 (25), 11889-11893.

42. (a) Kuehn, M.; Ogg, D.; Kihlberg, J.; Slonim, L.; Flemmer, K.; Bergfors, T.; Hultgren, S., Structural basis of pilus subunit recognition by the PapD chaperone. Science 1993, 262 (5137), 1234-1241; (b) Soto, G. E.; Dodson, K. W.; Ogg, D.; Liu, C.; Heuser, J.; Knight, S.; Kihlberg, J.; Jones, C. H.; Hultgren, S. J., Periplasmic chaperone recognition motif of subunits mediates quaternary interactions in the pilus. European Molecular Biology Organization Journal 1998, 17 (21), 6155-6167.

43. Chorell, E.; Pinkner, J. S.; Bengtsson, C.; Banchelin, T. S.-L.; Edvinsson, S.; Linusson, A.; Hultgren, S. J.; Almqvist, F., Mapping pilicide anti-virulence effect in Escherichia coli, a comprehensive structure–activity study. Bioorganic & Medicinal Chemistry 2012, 20 (9), 3128-3142.

44. G. Huber Trottman, R. J.-R., S. Kolczewski, R. D. Norcross, T. J. Woltering Use of Bi- and Tricyclic Pyridone Derivatives Against Alzheimer's Disease. WO 98/25930, 1998.

45. Åberg, V.; Norman, F.; Chorell, E.; Westermark, A.; Olofsson, A.; Sauer-Eriksson, A. E.; Almqvist, F., Microwave-assisted decarboxylation of bicyclic 2-pyridone scaffolds and identification of Aβ-peptide aggregation inhibitors. Organic & Biomolecular Chemistry 2005, 3 (15), 2817-2823.

46. Magano, J.; Dunetz, J. R., Large-Scale Applications of Transition Metal-Catalyzed Couplings for the Synthesis of Pharmaceuticals. Chemical Reviews 2011, 111 (3), 2177-2250.

47. (a) Ennis, D. S.; McManus, J.; Wood-Kaczmar, W.; Richardson, J.; Smith, G. E.; Carstairs, A., Multikilogram-Scale Synthesis of a Biphenyl Carboxylic Acid Derivative Using a Pd/C- Mediated Suzuki Coupling Approach. Organic Process Research & Development 1999, 3 (4), 248-252; (b) Glasnov, T. N.; Kappe, C. O., Toward a Continuous-Flow Synthesis of Boscalid®. Advanced Synthesis & Catalysis 2010, 352 (17), 3089-3097; (c) Kotha, S.; Lahiri, K.; Kashinath, D., Recent applications of the Suzuki–Miyaura cross-coupling reaction in organic synthesis. Tetrahedron 2002, 58 (48), 9633-9695; (d) Miller, W. D.; Fray, A. H.; Quatroche, J. T.; Sturgill, C. D., Suppression of a Palladium-Mediated Homocoupling in a Suzuki Cross-Coupling Reaction. Development of an Impurity Control Strategy Supporting Synthesis of LY451395. Organic Process Research & Development 2007, 11 (3), 359-364; (e) Ripin, D. H. B.; Bourassa, D. E.; Brandt, T.; Castaldi, M. J.; Frost, H. N.; Hawkins, J.;

Johnson, P. J.; Massett, S. S.; Neumann, K.; Phillips, J.; Raggon, J. W.; Rose, P. R.; Rutherford, J. L.; Sitter, B.; Stewart, A. M.; Vetelino, M. G.; Wei, L., Evaluation of Kilogram-Scale Sonagashira, Suzuki, and Heck Coupling Routes to Oncology Candidate CP-724,714. Organic Process Research & Development 2005, 9 (4), 440-450.

48. (a) Altemöller, M.; Podlech, J.; Fenske, D., Total Synthesis of Altenuene and Isoaltenuene. European Journal of Organic Chemistry 2006, 2006 (7), 1678-1684; (b) Johnson, P. D.; Sohn, J.-H.; Rawal, V. H., Synthesis of C-15 Vindoline Analogues by Palladium-Catalyzed Cross-Coupling Reactions. The Journal of Organic Chemistry 2006, 71 (20), 7899-7902; (c) Lee, S. J.; Gray, K. C.; Paek, J. S.; Burke, M. D., Simple, Efficient, and Modular Syntheses of Polyene Natural Products via Iterative Cross-Coupling. Journal of the American Chemical Society 2007, 130 (2), 466-468; (d) Lépine, R.; Zhu, J., Microwave-Assisted Intramolecular Suzuki−Miyaura Reaction to Macrocycle, a Concise Asymmetric Total Synthesis of Biphenomycin B. Organic Letters 2005, 7 (14), 2981-2984; (e) Wu, B.; Liu, Q.; Sulikowski, G. A., Total Synthesis of Apoptolidinone. Angewandte Chemie International Edition 2004, 43 (48), 6673- 6675.

49. Miyaura, N.; Suzuki, A., Palladium-Catalyzed Cross- Coupling Reactions of Organoboron Compounds. Chemical Reviews 1995, 95 (7), 2457-2483.

50. (a) Kirchhoff, J. H.; Dai, C.; Fu, G. C., A Method for Palladium-Catalyzed Cross-Couplings of Simple Alkyl Chlorides: Suzuki Reactions Catalyzed by [Pd2(dba)3]/PCy3. Angewandte Chemie International Edition 2002, 41 (11), 1945-1947; (b) Kirchhoff, J. H.; Netherton, M. R.; Hills, I. D.; Fu, G. C., Boronic Acids: New Coupling Partners in Room-Temperature Suzuki Reactions of Alkyl Bromides. Crystallographic Characterization of an Oxidative-Addition Adduct Generated under Remarkably Mild Conditions. Journal of the American Chemical Society 2002, 124 (46), 13662-13663; (c) Netherton, M. R.; Dai, C.; Neuschütz, K.; Fu, G. C., Room-Temperature Alkyl−Alkyl Suzuki Cross-Coupling of Alkyl Bromides that Possess β Hydrogens. Journal of the American Chemical Society 2001, 123 (41), 10099-10100; (d) Netherton, M. R.; Fu, G. C., Suzuki Cross-Couplings of Alkyl Tosylates that Possess β Hydrogen Atoms: Synthetic and Mechanistic Studies. Angewandte Chemie International Edition 2002, 41 (20), 3910-3912.

51. Gavara, L.; Boisse, T.; Rigo, B.; Hénichart, J.-P., A new method of bromination of aromatic rings by an iso-amyl nitrite/HBr system. Tetrahedron 2008, 64 (22), 4999-5004.

52. Bengtsson, C.; Almqvist, F., Regioselective Halogenations and Subsequent Suzuki−Miyaura Coupling onto Bicyclic 2-Pyridones. The Journal of Organic Chemistry 2009, 75 (3), 972-975.

53. Conreaux, D.; Bossharth, E.; Monteiro, N.; Desbordes, P.; Vors, J.-P.; Balme, G., Flexible Strategy for Differentially 3,5- Disubstituted 4-Oxypyridin-2(1H)-ones Based on Site-Selective Pd- Catalyzed Cross-Coupling Reactions. Organic Letters 2007, 9 (2), 271-274.

54. Seger, H.; Geyer, A., Synthesis and Diversification of Pyridone Dipeptide Chromophores. Synthesis 2006, 3224-3230.

55. Bengtsson, C.; Lindgren, A. E. G.; Uvell, H.; Almqvist, F., Design, synthesis and evaluation of triazole functionalized ring- fused 2-pyridones as antibacterial agents. European Journal of Medicinal Chemistry 2012, 54, 637-646.

56. (a) Bodurow, C.; Carr, M. A.; Moore, L. L., 2,2-dimethyl- 6-[(triphenylphosphoranylidene)methyl]-4h-1,3-dioxin-4-one. A four-carbon homologating agent requiring no activation. Organic Preparations and Procedures International 1990, 22 (1), 109-111; (b) Sato, M.; Sakaki, J.-i.; Takayama, K.; Kobayashi, S.; Suzuki, M.; Kaneko, C., A Novel Synthetic Method for Tetronic Acids from 1, 3- Dioxin-4-ones via Intra- or Intermolecular Ketene Trapping. Chemical & Pharmaceutical Bulletin 1990, 38 (1), 94-98.

57. Jones, R. C. F.; Tankard, M., A new sequence for the synthesis of 3-(poly)enoyltetramic acids. Journal of the Chemical Society, Perkin Transactions 1 1991, 0 (1), 240-241.

58. Cheruku, P.; Paptchikhine, A.; Ali, M.; Neudorfl, J.-M.; Andersson, P. G., Development of new thiazole-based iridium catalysts and their applications in the asymmetric hydrogenation of trisubstituted olefins. Organic & Biomolecular Chemistry 2008, 6 (2), 366-373.

59. Kharasch, M. S.; Sternfeld, E.; Mayo, F. R., The Peroxide Effect in the Rearrangement of α-Bromoacetoacetic Ester. Journal of the American Chemical Society 1937, 59 (9), 1655-1657.

60. Chorell, E.; Pinkner, J. S.; Phan, G.; Edvinsson, S.; Buelens, F.; Remaut, H.; Waksman, G.; Hultgren, S. J.; Almqvist, F., Design and Synthesis of C-2 Substituted Thiazolo and Dihydrothiazolo Ring-Fused 2-Pyridones: Pilicides with Increased Antivirulence Activity. Journal of Medicinal Chemistry 2010, 53 (15), 5690-5695.

61. Sellstedt, M.; Krishna Prasad, G.; Syam Krishnan, K.; Almqvist, F., Directed diversity-oriented synthesis. Ring-fused 5- to 10-membered rings from a common peptidomimetic 2-pyridone precursor. Tetrahedron Letters 2012, 53 (45), 6022-6024.

62. (a) Gonçalves, M. S. T., Fluorescent Labeling of Biomolecules with Organic Probes. Chemical Reviews 2009, 109 (1), 190-212; (b) Lavis, L. D.; Raines, R. T., Bright Ideas for Chemical Biology. ACS Chemical Biology 2008, 3 (3), 142-155; (c) Loudet, A.; Burgess, K., BODIPY Dyes and Their Derivatives: Syntheses and Spectroscopic Properties. Chemical Reviews 2007, 107 (11), 4891- 4932.

63. Chorell, E.; Bengtsson, C.; Sainte-Luce Banchelin, T.; Das, P.; Uvell, H.; Sinha, A. K.; Pinkner, J. S.; Hultgren, S. J.; Almqvist, F., Synthesis and application of a bromomethyl substituted scaffold to be used for efficient optimization of anti-virulence activity. European Journal of Medicinal Chemistry 2011, 46 (4), 1103-1116.

64. Mattsson, S.; Dahlström, M.; Karlsson, S., A mild hydrolysis of esters mediated by lithium salts. Tetrahedron Letters 2007, 48 (14), 2497-2499.

65. Raman, P.; Razavi, H.; Kelly, J. W., Titanium(IV)- Mediated Tandem Deprotection−Cyclodehydration of Protected Cysteine N-Amides: Biomimetic Syntheses of Thiazoline- and Thiazole-Containing Heterocycles. Organic Letters 2000, 2 (21), 3289-3292.

66. Wang, D.; Fan, J.; Gao, X.; Wang, B.; Sun, S.; Peng, X., Carboxyl BODIPY Dyes from Bicarboxylic Anhydrides: One-Pot Preparation, Spectral Properties, Photostability, and Biolabeling. The Journal of Organic Chemistry 2009, 74 (20), 7675-7683.

67. (a) Chorell, E.; Edvinsson, S.; Almqvist, F., Improved procedure for the enantioselective synthesis of dihydrooxazolo and dihydrothiazolo ring-fused 2-pyridones. Tetrahedron Letters 2010, 51 (18), 2461-2463; (b) Pemberton, N.; Pinkner, J. S.; Edvinsson, S.; Hultgren, S. J.; Almqvist, F., Synthesis and evaluation of dihydroimidazolo and dihydrooxazolo ring-fused 2-pyridones— targeting pilus biogenesis in uropathogenic bacteria. Tetrahedron 2008, 64 (40), 9368-9376.

68. Chorell, E.; Pinkner, J. S.; Bengtsson, C.; Edvinsson, S.; Cusumano, C. K.; Rosenbaum, E.; Johansson, L. B. Å.; Hultgren, S. J.; Almqvist, F., Design and Synthesis of Fluorescent Pilicides and Curlicides: Bioactive Tools to Study Bacterial Virulence Mechanisms. Chemistry – A European Journal 2012, 18 (15), 4522-4532.

69. (a) Appendino, G.; Bacchiega, S.; Minassi, A.; Cascio, M. G.; De Petrocellis, L.; Di Marzo, V., The 1,2,3-Triazole Ring as a Peptido- and Olefinomimetic Element: Discovery of Click Vanilloids and Cannabinoids. Angewandte Chemie 2007, 119 (48), 9472-9475; (b) Bock, V. D.; Speijer, D.; Hiemstra, H.; van Maarseveen, J. H., 1,2,3-Triazoles as peptide bond isosteres: synthesis and biological evaluation of cyclotetrapeptide mimics. Organic & Biomolecular Chemistry 2007, 5 (6), 971-975; (c) Brik, A.; Alexandratos, J.; Lin, Y.-C.; Elder, J. H.; Olson, A. J.; Wlodawer, A.; Goodsell, D. S.; Wong, C.-H., 1,2,3-Triazole as a Peptide Surrogate in the Rapid Synthesis of HIV-1 Protease Inhibitors. ChemBioChem 2005, 6 (7), 1167-1169; (d) Brik, A.; Muldoon, J.; Lin, Y.-C.; Elder, J. H.; Goodsell, D. S.; Olson, A. J.; Fokin, V. V.; Sharpless, K. B.; Wong, C.-H., Rapid Diversity- Oriented Synthesis in Microtiter Plates for In Situ Screening of HIV Protease Inhibitors. ChemBioChem 2003, 4 (11), 1246-1248; (e) Kim, S.; Cho, M.; Lee, T.; Lee, S.; Min, H.-Y.; Lee, S. K., Design, synthesis, and preliminary biological evaluation of a novel triazole analogue of ceramide. Bioorganic & Medicinal Chemistry Letters 2007, 17 (16), 4584-4587; (f) Lee, T.; Cho, M.; Ko, S.-Y.; Youn, H.-J.; Baek, D. J.; Cho, W.-J.; Kang, C.-Y.; Kim, S., Synthesis and Evaluation of 1,2,3- Triazole Containing Analogues of the Immunostimulant α-GalCer. Journal of Medicinal Chemistry 2007, 50 (3), 585-589; (g) Maurya, S. K.; Gollapalli, D. R.; Kirubakaran, S.; Zhang, M.; Johnson, C. R.; Benjamin, N. N.; Hedstrom, L.; Cuny, G. D., Triazole Inhibitors of Cryptosporidium parvum Inosine 5ʹ-Monophosphate Dehydrogenase. Journal of Medicinal Chemistry 2009, 52 (15), 4623-4630.

70. (a) Odlo, K.; Fournier-Dit-Chabert, J.; Ducki, S.; Gani, O. A. B. S. M.; Sylte, I.; Hansen, T. V., 1,2,3-Triazole analogs of

combretastatin A-4 as potential microtubule-binding agents. Bioorganic & Medicinal Chemistry 2010, 18 (18), 6874-6885; (b) Pagliai, F.; Pirali, T.; Del Grosso, E.; Di Brisco, R.; Tron, G. C.; Sorba, G.; Genazzani, A. A., Rapid Synthesis of Triazole-Modified Resveratrol Analogues via Click Chemistry. Journal of Medicinal Chemistry 2006, 49 (2), 467-470.

71. Yang, L.; Xu, W.; Chen, F.; Liu-Chen, L.-Y.; Ma, Z.; Lee, D. Y. W., Synthesis and biological evaluation of C-12 triazole and oxadiazole analogs of salvinorin A. Bioorganic & Medicinal Chemistry Letters 2009, 19 (5), 1301-1304.

72. Huisgen, R., Proceedings of the Chemical Society. Proceedings of the Chemical Society 1961, 357-396.

73. (a) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B., A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective “Ligation” of Azides and Terminal Alkynes. Angewandte Chemie International Edition 2002, 41 (14), 2596-2599; (b) Tornøe, C. W.; Christensen, C.; Meldal, M., Peptidotriazoles on Solid Phase: [1,2,3]-Triazoles by Regiospecific Copper(I)-Catalyzed 1,3-Dipolar Cycloadditions of Terminal Alkynes to Azides. The Journal of Organic Chemistry 2002, 67 (9), 3057- 3064.

74. Zhang, L.; Chen, X.; Xue, P.; Sun, H. H. Y.; Williams, I. D.; Sharpless, K. B.; Fokin, V. V.; Jia, G., Ruthenium-Catalyzed Cycloaddition of Alkynes and Organic Azides. Journal of the American Chemical Society 2005, 127 (46), 15998-15999.

75. (a) Meldal, M.; Tornøe, C. W., Cu-Catalyzed Azide−Alkyne Cycloaddition. Chemical Reviews 2008, 108 (8), 2952-3015; (b) Moses, J. E.; Moorhouse, A. D., The growing applications of click chemistry. Chemical Society Reviews 2007, 36 (8), 1249-1262; (c) Thirumurugan, P.; Matosiuk, D.; Jozwiak, K., Click Chemistry for Drug Development and Diverse Chemical– Biology Applications. Chemical Reviews 2013, DOI: 10.1021/cr200409f.

76. Bengtsson, C.; Almqvist, F., A Selective Intramolecular 5-exo-dig or 6-endo-dig Cyclization en Route to 2-Furanone or 2- Pyrone Containing Tricyclic Scaffolds. The Journal of Organic Chemistry 2011, 76 (23), 9817-9825.

77. Klein, M.; Krainz, K.; Redwan, I. N.; Dinér, P.; Grøtli, M., Synthesis of Chiral 1,4-Disubstituted-1,2,3-Triazole Derivatives from Amino Acids. Molecules 2009, 14 (12), 5124-5143.

78. (a) Horvath, I.; Sellstedt, M.; Weise, C.; Nordvall, L.-M.; Krishna Prasad, G.; Olofsson, A.; Larsson, G.; Almqvist, F.; Wittung- Stafshede, P., Modulation of α-synuclein fibrillization by ring-fused 2-pyridones: Templation and inhibition involve oligomers with different structure. Archives of Biochemistry and Biophysics 2013, 532 (2), 84-90; (b) Horvath, I.; Weise, C. F.; Andersson, E. K.; Chorell, E.; Sellstedt, M.; Bengtsson, C.; Olofsson, A.; Hultgren, S. J.; Chapman, M.; Wolf-Watz, M.; Almqvist, F.; Wittung-Stafshede, P., Mechanisms of Protein Oligomerization: Inhibitor of Functional Amyloids Templates α-Synuclein Fibrillation. Journal of the American Chemical Society 2012, 134 (7), 3439-3444.

79. Sellstedt, M.; Almqvist, F., A Novel Heterocyclic Scaffold Formed by Ring Expansion of a Cyclic Sulfone to Sulfonamides. Organic Letters 2009, 11 (23), 5470-5472.

80. Kwan, J. C.; Ratnayake, R.; Abboud, K. A.; Paul, V. J.; Luesch, H., Grassypeptolides A−C, Cytotoxic Bis-thiazoline Containing Marine Cyclodepsipeptides. The Journal of Organic Chemistry 2010, 75 (23), 8012-8023.

81. Lin, Z.; Antemano, R. R.; Hughen, R. W.; Tianero, M. D. B.; Peraud, O.; Haygood, M. G.; Concepcion, G. P.; Olivera, B. M.; Light, A.; Schmidt, E. W., Pulicatins A−E, Neuroactive Thiazoline Metabolites from Cone Snail-Associated Bacteria. Journal of Natural Products 2010, 73 (11), 1922-1926.

82. (a) Ino, A.; Hasegawa, Y.; Murabayashi, A., Synthetic studies of thiazoline and thiazolidine-containing natural products — 2. Total synthesis of the antimycoplasma antibiotic micacocidin. Tetrahedron 1999, 55 (34), 10283-10294; (b) Ino, A.; Murabayashi, A., Synthetic studies of thiazoline and thiazolidine-containing natural products — 1. Phosphorus pentachloride-mediated thiazoline construction reaction. Tetrahedron 1999, 55 (34), 10271-10282; (c) Ino, A.; Murabayashi, A., Synthetic studies of thiazoline and thiazolidine-containing natural products. Part 3: Total synthesis and absolute configuration of the siderophore yersiniabactin. Tetrahedron 2001, 57 (10), 1897-1902.

83. (a) Brechbühl, J.; Moine, F.; Klaey, M.; Nenniger- Tosato, M.; Hurni, N.; Sporkert, F.; Giroud, C.; Broillet, M.-C., Mouse alarm pheromone shares structural similarity with predator scents. Proceedings of the National Academy of Sciences 2013, 110 (12), 4762-4767; (b) Sharrow, S. D.; Novotny, M. V.; Stone, M. J., Thermodynamic Analysis of Binding between Mouse Major Urinary Protein-I and the Pheromone 2-sec-Butyl-4,5-dihydrothiazole†. Biochemistry 2003, 42 (20), 6302-6309; (c) Tashiro, T.; Mori, K., Synthesis of the Enantiomers of 2-sec-Butyl-4,5-dihydrothiazole and (1R,5S,7R)-3,4-Dehydro-exo-brevicomin, Pheromone Components of the Male Mouse, Mus musculus. European Journal of Organic Chemistry 1999, 1999 (9), 2167-2173; (d) Timm, D. E.; Baker, L. J.; Mueller, H.; Zidek, L.; Novotny, M. V., Structural basis of pheromone binding to mouse major urinary protein (MUP-I). Protein Science 2001, 10 (5), 997-1004.

84. (a) Adams, A.; De Kimpe, N., Chemistry of 2-Acetyl-1- pyrroline, 6-Acetyl-1,2,3,4-tetrahydropyridine, 2-Acetyl-2-thiazoline, and 5-Acetyl-2,3-dihydro-4H-thiazine: Extraordinary Maillard Flavor Compounds. Chemical Reviews 2006, 106 (6), 2299-2319; (b) Bel Rhlid, R.; Fleury, Y.; Blank, I.; Fay, L. B.; Welti, D. H.; Arce Vera, F.; Juillerat, M. A., Generation of Roasted Notes Based on 2-Acetyl-2- thiazoline and Its Precursor, 2-(1-Hydroxyethyl)-4,5-dihydrothiazole, by Combined Bio and Thermal Approaches. Journal of Agricultural and Food Chemistry 2002, 50 (8), 2350-2355.

85. (a) Abrunhosa, I.; Delain-Bioton, L.; Gaumont, A.-C.; Gulea, M.; Masson, S., Chiral thiazoline ligands: application in Pd- catalysed allylic substitution. Tetrahedron 2004, 60 (41), 9263- 9272; (b) Abrunhosa, I.; Gulea, M.; Levillain, J.; Masson, S., Synthesis of new chiral thiazoline-containing ligands. Tetrahedron: Asymmetry 2001, 12 (20), 2851-2859; (c) Gaumont, A.-C.; Gulea, M.; Levillain, J., Overview of the Chemistry of 2-Thiazolines. Chemical Reviews 2009, 109 (3), 1371-1401.

86. (a) Nishio, T.; Kodama, Y.; Tsurumi, Y., Synthesis of Chiral Bis-Thiazolines and Asymmetric Diels–Alder Reactions. Phosphorus, Sulfur, and Silicon and the Related Elements 2005, 180 (5-6), 1449-1450; (b) Yamakuchi, M.; Matsunaga, H.; Tokuda, R.; Ishizuka, T.; Nakajima, M.; Kunieda, T., Sterically congested ‘roofed’ 2-thiazolines as new chiral ligands for copper(II)-catalyzed asymmetric Diels–Alder reactions. Tetrahedron Letters 2005, 46 (23), 4019-4022.

87. (a) Gong, Z.; Liu, Q.; Xue, P.; Li, K.; Song, Z.; Liu, Z.; Jin, Y., Novel chiral thiazoline-containing N―O ligands in the asymmetric addition of diethylzinc to aldehydes: substituent effect on the enantioselectivity. Applied Organometallic Chemistry 2012, 26 (3), 121-129; (b) Wipf, P.; Wang, X., A New Ligand Scaffold for Catalytic Asymmetric Alkylzinc Additions to Aldehydes. Organic Letters 2002, 4 (7), 1197-1200.

88. Jia, Y.; Yang, W.; Du, D.-M., Asymmetric Friedel-Crafts alkylation of indoles with 3-nitro-2H-chromenes catalyzed by diphenylamine-linked bis(oxazoline) and bis(thiazoline) Zn(II) complexes. Organic & Biomolecular Chemistry 2012, 10 (24), 4739- 4746.

89. (a) Meyers, A. I.; Durandetta, J. L., 2-Thiazolines in organic synthesis. Synthesis of mono-, di-, and trialkylacetaldehydes. The Journal of Organic Chemistry 1975, 40 (14), 2021-2025; (b) Meyers, A. I.; Durandetta, J. L.; Munavu, R., 2-Thiazolines in organic synthesis. Formation of .beta.-hydroxy aldehydes with protected hydroxy groups. Synthesis of homoallylic alcohols. The Journal of Organic Chemistry 1975, 40 (14), 2025-2029; (c) Meyers, A. I.; Tavares, F. X., Oxidation of Oxazolines and Thiazolines to Oxazoles and Thiazoles. Application of the Kharasch−Sosnovsky Reaction. The Journal of Organic Chemistry 1996, 61 (23), 8207-8215.

90. Tao, B.; Schlingloff, G.; Sharpless, K. B., Reversal of regioselection in the asymmetric aminohydroxylation of cinnamates. Tetrahedron Letters 1998, 39 (17), 2507-2510.

91. (a) Galéotti, N.; Montagne, C.; Poncet, J.; Jouin, P., Formation of oxazolines and thiazolines in peptides by the mitsunobu reaction. Tetrahedron Letters 1992, 33 (20), 2807-2810; (b) Lafargue, P.; Guenot, P.; Lellouche, J.-P., Preparation of 2- Thiazolines from (1,2)-Thioamido-Alcohols; DAST as a Useful Reagent. Synlett 1995, 2, 171-172; (c) Ori, M.; Nishio, T., Thionation of N-Acylthreonine and Its Methyl Ester with Lawesson's Reagent: Synthesis of 5-Oxazolones, 5-Thiazolones and Thiazolines. Heterocycles 2001, 54 (1), 201-208; (d) Wipf, P.; Miller, C. P., An investigation of the Mitsunobu reaction in the preparation of peptide oxazolines, thiazolines, and aziridines. Tetrahedron Letters 1992, 33 (42), 6267-6270.

92. Lu, X.; Xu, Z.; Yang, G., Process Development of the Sharpless Catalytic Asymmetric Dihydroxylation Reaction To Prepare

Methyl (2R,3S)-2,3-Dihydroxy-3-phenylpropionate. Organic Process Research & Development 2000, 4 (6), 575-576.

93. Nicolaou, K. C.; Dethe, D. H.; Leung, G. Y. C.; Zou, B.; Chen, D. Y. K., Total Synthesis of Thiopeptide Antibiotics GE2270A, GE2270T, and GE2270C1. Chemistry – An Asian Journal 2008, 3 (2), 413-429.

94. Fleming, P. R.; Sharpless, K. B., Selective transformations of threo-2,3-dihydroxy esters. The Journal of Organic Chemistry 1991, 56 (8), 2869-2875.

95. (a) Nishio, T., Sulfur-containing heterocycles derived by the reaction of hydroxy-amides and Lawesson's reagent. Tetrahedron Letters 1995, 36 (34), 6113-6116; (b) Nishio, T., Reaction of (1,ω)-N- Acylamino Alcohols with Lawesson's Reagent: Synthesis of Sulfur- Containing Heterocycles. The Journal of Organic Chemistry 1997, 62 (4), 1106-1111; (c) Tárraga, A.; Molina, P.; Curiel, D.; Bautista, D., Asymmetric synthesis of ferrocenylazido alcohols and their derivatization to novel chiral ferrocenyl-thiazoline ligands with C2- symmetry. Tetrahedron: Asymmetry 2002, 13 (15), 1621-1628.

96. Neises, B.; Steglich, W., Simple Method for the Esterification of Carboxylic Acids. Angewandte Chemie International Edition in English 1978, 17 (7), 522-524.

97. (a) Nishio, T., Direct conversion of alcohols into thiols. Journal of the Chemical Society, Perkin Transactions 1 1993, 0 (10), 1113-1117; (b) Nishio, T.; Konno, Y.; Ori, M.; Sakamoto, M., Stereoselective Synthesis of 4H-5,6-Dihydro-1,3-thiazines by the Reaction of 3-N-Acylamino Alcohols with Lawesson’s Reagent. European Journal of Organic Chemistry 2001, 2001 (18), 3553- 3557.

98. Miyata, O.; Shinada, T.; Ninomiya, I.; Naito, T.; Date, T.; Okamura, K.; Inagaki, S., Stereospecific nucleophilic addition reactions to olefins. Addition of thiols to .alpha.,.beta.-unsaturated carboxylic acid derivatives. The Journal of Organic Chemistry 1991, 56 (23), 6556-6564.

99. (a) Baggs, M. E. M.; Gregory, B., Intramolecular Michael-type additions. II. Synthesis of derivatives of 7-thia-2- azabicyclo[3.2.1]oct-3-ene, a novel heterocyclic ring system. Canadian Journal of Chemistry 1980, 58 (8), 794-802; (b) Baldwin,

J. E.; Cutting, J.; Dupont, W.; Kruse, L.; Silberman, L.; Thomas, R. C., 5-Endo-trigonal reactions: a disfavoured ring closure. Journal of the Chemical Society, Chemical Communications 1976, 0 (18), 736-738.

100. Sellstedt, M.; Almqvist, F., A Three-Component Reaction Forming Naphthyridones – Synthesis of Lophocladine Analogs. Organic Letters 2011, 13 (19), 5278-5281.

101. (a) Hellal, M.; Bourguignon, J.-J.; Bihel, F. J. J., 6-endo- dig Cyclization of heteroarylesters to alkynes promoted by Lewis acid catalyst in the presence of Brønsted acid. Tetrahedron Letters 2008, 49 (1), 62-65; (b) Uchiyama, M.; Ozawa, H.; Takuma, K.; Matsumoto, Y.; Yonehara, M.; Hiroya, K.; Sakamoto, T., Regiocontrolled Intramolecular Cyclizations of Carboxylic Acids to Carbon−Carbon Triple Bonds Promoted by Acid or Base Catalyst. Organic Letters 2006, 8 (24), 5517-5520; (c) Yao, T.; Larock, R. C., Synthesis of Isocoumarins and α-Pyrones via Electrophilic Cyclization. The Journal of Organic Chemistry 2003, 68 (15), 5936-5942.

102. Serrano-Wu, M. H.; Regueiro-Ren, A.; St. Laurent, D. R.; Carroll, T. M.; Balasubramanian, B. N., Mild deprotection of 2- (trimethylsilyl)ethyl esters. Tetrahedron Letters 2001, 42 (49), 8593-8595.

103. Jia, C.; Kitamura, T.; Fujiwara, Y., Catalytic Functionalization of Arenes and Alkanes via C−H Bond Activation. Accounts of Chemical Research 2001, 34 (8), 633-639.

104. de Meijere, A., Bonding Properties of Cyclopropane and Their Chemical Consequences. Angewandte Chemie International Edition in English 1979, 18 (11), 809-826.