Photoredox Catalyzed Regioselective Phosphonylation of Ring Fused 2- Pyridones and Their Biological Evaluation

Photoredox Catalyzed Regioselective Phosphonylation of Ring Fused 2- Pyridones and their Biological Evaluation

Amandeep Kaur

Master thesis: 60 ECTS Supervisor: Prof. Fredrik Almqvist Examiner: Passed:

Abstract

Synthetic methodology for the C-6 phosphonylation of biologically active thiazolino- 2-pyridones under photoredox conditions has been developed. The photoredox method utilizes (Ir[dF(CF3)ppy]2(dtbpy))PF6 as photocatalyst, ceric ammonium nitrate as an oxidant, and trialkyl phosphites as phosphorus source to provide phosphonylated 2- pyridones under very mild reaction conditions. The phosphonate esters could be further hydrolyzed to the corresponding phosphonic acids to investigate structure activity relationships.

R1 R1 R1 R R R 2 S C-6 Phosphonylation 2 S Hydrolysis 2 S N N N (EtO) OP R3 2 R3 (HO)2OP R3 O O O O O O O O O

List of abbreviations

AcCL Acetyl chloride aq. Aqueous BrTMS Bromotrimethylsilane calcd. Calculated CAN Ceric ammonium nitrate DCA 9,10-Anthracenedicarbonitrile DCC N,N'-Dicyclohexylcarbodiimide DCE Dichloroethane DCM Dichloromethane DIPEA N,N-Diisopropylethylamine DMAP 4-Dimethylaminopyridine DMF Dimethylformamide DMSO Dimethyl sulfoxide Et2O Diethyl ether EtOAc Ethyl acetate EtOH Ethanol eq. Equivalent(s) HATU 1-[Bis(dimethylamino)methylene]-1H- 1,2,3 triazolo[4,5-b] pyridinium 3-oxide HPLC High-performance liquid chromatography HRMS High resolution mass spectrometry Hrs Hours (Ir[dF(CF3)ppy]2(dtbpy))PF6 2,2ʹ-bipyridine]bis[3,5-difluoro-2-[5-(- (trifluoromethyl-2- pyridinyl]phenyl]iridium(III) hexafluorophosphate LCMS Liquid chromatography–mass spectrometry Me Methyl MeCN Acetonitrile MeOH Methanol MWI Microwave Irradiation NMR Nuclear Magnetic Resonance OAc Acetate Ph Phenyl PhMe Toluene HPO(OEt)2 Diethyl phosphite P(OEt)3 Triethyl phosphite Rt Room temperature sat. Saturated SET Single electron transfer UPEC Uropathogenic Escherichia coli TBAF Tetrabutylammonium fluoride TEA Triethylamine TFA Trifluoroacetic acid TLC Thin layer chromatography ThT Thioflavin T

Rh6G Rhodamine 6G

Author contribution

Design of the synthetic pathways, synthesis and purification of all the intermediates and final products, collection of all the analytical data and interpretation of the results.

III

Table of contents

Abstract………………………………………………………………………………...I List of Abbreviation…………………………………………………………………...II Author´s contribution………………………………………………………………...III 1. Introduction………………………………………………………………………..1 1.1 Thiazolino-2-Pyridones………………………………………………………..1 1.2 Photoredox Catalysis…………………………………………………………..2 1.3 Photoredox Phosphonylation…………………………………………………..2 1.4 Aim of the diploma work……………………………………………………...4 2. Popular scientific summary………………………………………………………..4 3. Social and ethical aspects………………………………………………………….5 4. Results and Discussion…………………………………………………………….5 4.1 Synthesis of thiazolino-2-pyridones …………………………………………..5 4.2 Synthesis of Photocatalyst …………………………………………………….6 4.3 Phosphonylation of 2-Pyridones……………………………………………….6 4.4 Mechanism of the Phosphonylation Reaction…………………………………11 4.5 Biological Evaluation………………………………………………………….11 5. Conclusions and Outlook………………………………………………………….14 6. Experimental………………………………………………………………………15 6.1 General Information…………………………………………………………...15 6.2 General procedure for the synthesis of thiazolines 17a-c…………………….15 6.3 General procedure for the synthesis of Meldrum,s acid derivative 20a-c…….16 6.4 General procedure for the synthesis of thiazlino-2-pyridones 21a-e………….17 6.5 General procedure for the synthesis of amides 29a-c………………………….18 6.6 General procedure for the synthesis of compounds 27a-d and 30a-c…………19 6.7General procedure for the synthesis of compounds 28a-d……………………..22 Acknowledgement……………………………………………………………………25 References……………………………………………………………………………26 Appendix 1…………………………………………………………………………...28 Appendix 2…………………………………………………………………………...46

1. Introduction

1.1 Thiazolino-2-pyridones

Thiazolino-2-pyridone scaffolds are designed peptidomimetics which are known to inhibit the formation of virulence factors such as pili in E. coli (UPEC), therefore named Pilicides (Compound 1, Figure 1).1 These bicyclic heterocycles mimic the C-terminal of a peptide and their substitution with bulky aryl groups result in the analogues (Compound 2, Figure 1) which inhibit the formation of bacterial amyloids known as curli and Aβ-peptides.2 Depending upon the substitution pattern some of the derivatives have been shown to inhibit L. monocytogenes by binding and attenuating the transcriptional regulator PrfA3(Compound 4, Figure 1) while others inhibit Mycobacterium Tuberculosis (Compound 3, Figure 1).4 Further, the replacement of carboxylic group at C-3 with a phenyl amide (Compound 5, Figure 1) or amide isosteres resulted in the compounds which inhibit Chlamydia trachomatis infectivity.5 Rewardingly, the introduction of an amine at the C-6 position (Compound 6, Figure 1) not only improved the inhibitory activity against C. trachomatis but also physicochemical properties.5 Further, the bicyclic framework has been extended to tricyclic/polycyclic scaffolds to enable them to modulate a-synuclein amyloid formation,6,7 which is associated with Parkinson's disease. For example, the benzoquinoline annulated polyheterocycles (Compound 7, Figure 1) inhibit the α-synuclein amyloid formation while pyridine fused tricyclic framework (Compound 8, Figure 1) binds to mature α-synuclein fibrils.

CF3

N S S S N N N CO H O CO2H O CO2H O 2 1 2 3

S S S N N N H2N O CO2H O N N O H O 4 5 6 O H

S S N N N N CO2H O CO2H O O2N 7 8

Figure 1. Examples of biologically active ring-fused thiazolino-2-pyridones

1.2 Photoredox Catalysis

Visible light photo-redox catalysis is one of the fastest growing fields in organic chemistry. Over the past century, light mediated catalysis has enabled the substitution of non-functional C-H bonds.7a-c In photo-redox catalysis, photonic energy can be converted into usable chemical energy by different modes, for example, energy transfer and single-electron transfer (SET). The main factor in the rapid success of photo-redox catalysis is the accessibility of metal polypyridyl complexes and organic dyes which upon excitation give access to reactive species via single-electron transfer (SET) by reacting with organic substrates.7a-c In these photo-redox reactions, the photocatalyst is excited by visible light, at wavelengths where reacting molecules do not absorb. The resultant excited photocatalyst can either act as a strong oxidant or strong reductant simultaneously.7a-c Below are the most commonly used catalysts.

F C 3 F PF6 PF6 N t-Bu N t-Bu N 2Cl NC CN N N N N N F Ir Ir Ru 6H2O F N N N N N N N N N t-Bu t-Bu N F F3C

(Ir[dF(CF3)ppy]2(dtbpy))PF6 Ir(ppy)2(dtbpy)PF6 Ru(bpy)3Cl2.6H2O 4CzIPN

CN O BF Br Br 4 BF4

HO O OH N O CN Br Br Me 2,4,6-Triphenylpyrylium 9-Mesityl-10-methylacridinium 1,2-Dicyanoanthracene (DCA) Eosin Y tetraﬂuoroborate tetraﬂuoroborate

Figure 2. Commonly used metal and organo-photocatalysts

1.3 Photoredox Phosphonylation

Phosphorous containing heterocycles are important classes of compounds with broad applications in synthetic organic,8 medicinal9 and material chemistry.10 Since phosphorous substituents play an important role in regulating the biological and material functions, considerable efforts have been made for the development of organo- phosphorous compounds in the past decades. The conventional methods use stoichiometric amounts of alkali metals for the formation of P-C bonds. These protocols are tedious, have narrow substrate scope and produce large quantities of waste. In the past several years, the transition-metal catalysis (i.e, Rh, Pd, Cu, Mn, Ni, and Ag based catalysts) have been widely utilized in coupling processes for the construction of Csp2/Csp3-Pbonds.12 The addition of phosphoryl radicals to olefins or arenes by using metal complexes and electrochemical methods have offered an alternate method for the preparation of organophosphorus compounds.13

However, these methods suffer from disadvantages like harsh reaction conditions and limited substrate scope. Visible-light-mediated photoredox catalysis has recently been used for the efficient phosphonylation of aryls/heteroaryls.14 Photo-redox phosphonylation strategies have advantages over conventional methods as stoichiometric amounts of radical initiators can be avoided.14 Moreover, photo-redox methods are mild and can tolerate a wide range of functional groups. Typically, phosphonylation reactions work by the addition of P-centered radicals to C-H bonds which can be generated via single electron transfer from a photocatalyst. Recently König reported the synthesis of aryl/heteroaryl phosphonates by using aryl/heteroaryl halides and trialkyl phosphite in the presence of rhodamine 6G as the photocatalyst.15 The reaction has a wide substrate scope where a range of aryl/heteroaryls were phosphonylated under mild conditions in good yields (Scheme 1). O P(OEt)3 OEt Br P Rh-6G, DIPEA OEt Ar Ar Blue LED, DMSO rt 9 10

O O OEt OEt P P OEt OEt

NC 80% 59%

O OEt P O OEt N P OEt NC S OEt 78% 69% Scheme 1

The direct phosphonylation of N-protected indole derivatives to form 2-phosphonylated 16 indoles was disclosed by An et al. The method used Ru(bpy)3(PF6)2 as photocatalyst and oxygen as the oxidant to produce phosphonylated products in moderate to good yields (Scheme 2). 1 O Ru(bpy)3 PF6 O R H R1 + P P Visible light 2 N 2 N R R CH Cl , rt, air 11 R 2 2 12 R Scheme 2

Very recently König developed direct visible-light photoredox C-H bond phosphonylation of electron-rich arenes and heteroarenes.17 The photoredox catalytic protocol utilizes electron-rich and biologically important arenes as substrates,

[Ru(bpz)3][PF6]2 as photocatalyst, ammonium persulfate as oxidant, and trialkyl phosphites as the phosphorus source to provide a wide range of aryl phosphonates at ambient temperature under very mild reaction conditions (Scheme 3) O P(OEt)3 OR P Ru(bpz)3, (NH4)S2O8 OR R R Blue LED, MeCN rt 13 14

OMe O OMe O OEt OEt P P OEt OEt

MeO MeO OMe OMe 99% 79%

OMe O O OEt OEt P P OEt OEt BocHN OMe 62% 63% Scheme 3

1.4 Aim of the diploma work

Thiazolino-2-pyridones are privileged scaffolds which can demonstrate different biological activities depending upon the substitution pattern.1-7 It has been shown that the introduction of polar groups, like an amine, significantly improved the biological activities and properties of bicyclic 2-pyridones.5 The aim of the present study is to develop synthetic methodology for the phosphonylation of 2-pyridones at the C-6 position under photoredox catalyzed conditions. We envisioned that phosphonylation may result in compounds with improved aqueous solubility, physicochemical properties and biological activities. In addition, a new efficient phosphonylation methodology can be of great importance to the medicinal chemistry community.

2. Popular Scientific Summary

In the past decades, chemists have invented several new metal/organic catalysts to make elegant molecules. Before the discovery of catalysts, molecules could be made by thermal reactions i.e. heating with acids and bases which sometime resulted in the decomposition of the reactants. The discovery of the catalysts (organocatalysis, photocatalysis, electro-catalysis, biocatalysis, etc.) has reduced the energy barriers, thus, making the reactions accessible which were previously impossible. The catalytic reactions can tolerate almost every functional group, and moreover inert C-H bonds can be functionalized. The most commonly used catalyzed reactions are the cross-couplings for the formation of carbon–carbon and carbon–heteroatom bonds, asymmetric hydrogenation, oxidation, asymmetric addition, and metathesis.18 In medicinal chemistry, there is always need of new synthetic methods to construct structural diverse molecules in an efficient manner. The catalyzed reactions have been extensively used in the preparation of libraries of pharmaceutical ingredients.18

Photochemistry is a relatively old field but energetically it is potentially a sustainable process to perform chemical reactions. Recently photoredox chemistry has made significant progress due to the discovery of multiple catalysts, ligands and reaction conditions. The important chemical transformations which previously often required multiple steps and/or protecting group manipulation, can be done efficiently in a single step under photoredox conditions.19 In medicinal chemistry, Suzuki Miyaura and other transition-metal catalyzed reactions are commonly used C-C bond forming strategies. 4

Photoredox chemistry has also found its applications in the formation of C-C bonds. The formation of Csp3–Csp2 bonds generally requires multiple steps, however, MacMillan’s group has recently developed methodology where protected amino acids upon reductive decarboxylation at the α-carbon forms a new bond with an aromatic ring in one step.20 This methodology has been utilized for the synthesis of Q203, which is in clinical trials for tuberculosis.20 Other important transformations in medicinal chemistry is the formation of C-X (X= O, N, S, P) hetero bonds.7c Although there are a large number of reactions available for the construction of C-X bonds, most of which are dominated by Buchwald-Hartwig and Chan-Lam couplings. The photo redox catalysis, provides an alternative and mild method to access C-X bonds where traditional methods fail.19

3. Social and Ethical Aspects

There are no ethical concerns regarding this research project. The target molecules and the synthesized intermediates will only be tested in vitro. Further testing of interesting molecules in vivo will be done in collaboration and ethical permits will be applied for if this comes into reality. The project was conducted according to the safety regulations of the Department of Chemistry at the Umeå University.

4. Results and Discussion 4.1 Synthesis of the thiazolino-2-pyridones

The bicyclic 2-pyridone required for the phosphonylation was prepared following the reported procedure6 as shown in Scheme 1. The first building block, Δ²-thiazoline 17 was synthesized in two steps by reacting nitrile (15) with acetyl chloride and ethanol to give ethyl acetimidate hydrochloride (16), which on reaction with L-cysteine methyl ester hydrochloride afforded 17. Acyl Meldrum’s derivative 20 as the second building block was synthesized by a condensation reaction of Meldrum’s acid (18) with the substituted carboxylic acid 19. Reacting the Δ²-thiazoline 17 with Meldrum’s acid derivative 20 by heating under microwave irradiation (MWI) and acidic conditions at 120 °C for 3 minutes resulted in the synthesis of desired thiazolo-2-pyridone scaffold 21a-e in good yields.

R1 Cl NH L-Cysteine methyl ester AcCl, EtOH 2 TEA S R1 CN R1 rt, 20 h O DCM, 0 °C -rt, 20 h N

15 16 17 CO2Me R1 TFA, DCE R2 S R2 MWI, 120 °C, N HO 3 min O CO2Me O O O OH Meldrum's acid 21a-e + DCC, DMAP O O O O 2 R DCM, 0 °C -rt, 20 h O O

18 19 20

CF3

O S S S N N N CO Me CO Me O 2 O 2 O CO2Me 21a; 76% 21b; 76% 21c; 80%

F3C S Cl S N N

O CO2Me O CO2Me 21d; 70% 21e; 78%

Scheme 1: Synthesis of Thiazolino ring-fused 2-pyridones

4.2 Synthesis of the photocatalyst

The photocatalyst (Ir[dF(CF3)ppy]2(dtbpy))PF6 26 which is expensive to buy was prepared according to the reported procedure.21 The synthetic route is given in scheme 5. In the first step, intermediate 24 was prepared by the Suzuki coupling of 2-chloro-4- methyl pyridine 22 with 2,4-difluorophenyl boronic acid 23 using sodium bicarbonate and Pd(PPh3)4 catalyst in a mixture of toluene/benzene. The reaction mixture was refluxed for 72 hours to give intermediate 24. In the next step, IrCl3 hydrate was treated with intermediate 24 by heating under MWI at 200 °C in ethylene glycol for 50 minutes followed by reaction with 4,4′-Di-tert-butyl-2,2′-dipyridyl 25 under the same condition as used for intermediate 24. Finally, the hexafluorophosphate salt of catalyst was prepared by treating with ammonium hexafluorophosphate. The desired product 26 was purified by recrystallization from acetone and diethyl ether (1:4) at 0o temperature.

B(OH)2 F

F3C F PF 23 6 F t-Bu N N 1) IrCl3.hydrate, ethylene glycol N Cl Pd(PPh) , Na CO o N 4 2 3 F C F MWI, 200 C, 50 min F 3 Ir F N F C Toluene, benzene, N N 3 F 2) 22 reﬂux, 72 h 24 t-Bu N 65% F t-Bu 25 t-Bu F3C 26 MWI, 200 oC, 50 min (Ir[dF(CF3)ppy]2(dtbpy))PF6 3) aq. NH4PF6 72% Scheme 5: Synthesis of Photocatalyst

4.3 Phosphonylation of 2-Pyridones

We began our photoredox phosphonylation protocol using 21a as test substrate and diethylphosphite as the coupling partner. We evaluated a range of photocatalysts in the presence of ceric ammonium nitrate and dibasic potassium phosphate in dry DMF. The 6

results are summarized in Table 1. The reactions were slow and only 10-20% conversion

was observed by LCMS in the case of (Ir[dF(CF3)ppy]2(dtbpy))PF6 (entry 1),

Ru(bpy)3Cl2.6H2O (entry 2), and DCA (entry 3). Notably, no desired product was observed after stirring for 18 hours in the case of Eosin Y (entry 4). The removal of base did not have any effect on the reaction outcome (entry 5 and 6). Next, we screened a range of oxidizing agents. Since, similar conversions were obtained in case of DCA and

(Ir[dF(CF3)ppy]2(dtbpy))PF6, we decided to continue with inexpensive DCA as photocatalyst. Unfortunately, only the starting material was recovered after 18 hours in case of all other tested oxidizing agents (entry 7-10). Table 1: Screening of photocatalysts, base and oxidants for the C-6 phosphonylation of 2-pyridones using diethyl phosphite.

F3C F3C

PC S S N N Oxidant, base, HPO(OEt)2 (EtO) OP DMF 2 O CO2Me O CO2Me 21a 27a

Entry Photocatalyst Base Oxidant LEDs Conversiona Time 1 (Ir[dF(CF3)ppy]2(dtbpy))PF6 K2HPO4 CAN 395 nm 10-20% 18

2 Ru(bpy)3Cl2.6H2O K2HPO4 CAN 455 nm 0-10% 18

3 DCA K2HPO4 CAN 455 nm 10-20% 18

4 EOSIN Y K2HPO4 CAN 529 nm - 18

5 (Ir[dF(CF3)ppy]2(dtbpy))PF6 - CAN 395 nm 10-20% 18 6 DCA - CAN 455 nm 10-20% 18

7 DCA K2HPO4 TBAP 455 nm - 18

8 DCA K2HPO4 K₂S₂O₈ 455 nm - 18

9 DCA K2HPO4 Air 455 nm - 18

10 DCA K2HPO4 MnO2 455 nm - 18

2-Pyridone 1a was treated with. of diethyl phosphite 2 (3.0 eq.), base (1.1eq), photocatalyst (2 mol%) and oxidant (1.5 eq). in DMF (0.1M). The reaction mixtures were stirred under light for 18 hours. aThe progress of reaction was determined by LCMS. Since,with other oxidizing agents no conversion had been taken place so CAN was chosen as oxidizing agent and we changed the phosphonylating agent to triethyl phosphite and again tested different photocatalysts in Dry DMF under respective LEDs

as summarized in Table 2. Gratifyingly, (Ir[dF(CF3)ppy]2(dtbpy))PF6 gave 80-90% (entry 1) while only 40-50% conversion was observed in case of DCA (entry 2) as

7 detemined by LCMS. None of the other tested photocatalysts resulted in the desired product. Table 2 : Screening of photocatalysts by replacing diethyl phosphite with triethyl phosphite.

F3C F3C

PC S S

N CAN, P(OEt)3, DMF N (EtO)2OP O CO2Me O CO2Me 21a 27a

Entry Photocatalyst LEDs Conversiona Time(hr) 1 Ir[dF(CF3)ppy]2(dtbpy))PF6 395 nm 80-90% 18

2 Ru(bpy)3Cl2.6H2O 450 nm 20-30% 18

3 DCA 450 nm 40-50% 18

4 Eosin y 529 nm - 18

5 9-Mesityl-10- 395 nm - 18 methylacridinium tetrafluoroborate 6 2,4,6-Triphenylpyrylium 455 nm - 18 tetrafluoroborate 7 4CzIPN 455 nm - 18 2-Pyridone 1a was treated with triethyl phosphite (5.0 eq.), CAN (1.5 eq) and photocatalyst (2mol%) in DMF (0.1M). The reaction mixtures were stirred under light for 18 hours. aThe progress of reaction was determined by LCMS. Finally, solvents were evaluated for the phosphonylation of thiazolino 2-pyridones. DMF and acetonitrile were found to be best while the other tested solvents did not give any conversion as determined by LCMS (Table 3). Since DMF has high boiling point and is difficult to remove during the work up, acetonitrile was chosen as solvent in further reactions. Table 3: Screening of solvents for the C-6 phosphonylation of 2-Pyridones.

F3C F3C

Ir[dF(CF3)ppy]2(dtbpy))PF6

S CAN, P(OEt)3 S N Blue LED (395 nm) N (EtO)2OP O CO2Me O CO2Me 21a 27a

Solvent Conversiona Time(hr) DMF 80-90% 18 DCM - 18 MeOH - 18 8

MeCN 100% 18 THF - 18 2-Pyridone 1a was treated with triethyl phosphite (3.0 eq.), CAN (1.0 eq) and (Ir[dF(CF3)ppy]2(dtbpy))PF6 (2mol%) in tested solvents (0.1M). The reaction mixture were stirred under blue LEDs (395 nm) for 18 hours. aThe progress of reaction was determined by LCMS. After improving the reaction conditions, we sought to investigate the scope of photoredox C-6 phosphonylation. As shown in Scheme 6, a range of variedly substituted 2-pyridones can be phosphonylated in good yield under improved conditions. However, the reaction did not work with 2-pyridones having a chloro methyl substituent at the C- 7 position. Unfortunately, it was also very difficult to separate the product from unreacted triethyl phosphite even after repeated column chromatography. Different strategies were used to remove the excess of triethyl phosphite without any success. Benzyl bromide has been known to form a salt with triethyl phosphite which can be removed by aqueous workup. Thus, benzyl bromide was added to the reaction mixture after the completion of the phosphonylation reaction and stirred at different temperatures (rt to 60 °C) for 3-24 hours. However, in our case addition of benzyl bromide resulted in the reduction of yield of the phosphonylated product without affecting the amount of triethylphosphite. Further, even the lyophilization of the phosphonylated products could not remove the triethyl phosphite. Therefore, all the phosphonylated products 27a-d were used as intermediates and reacted further as crude mixtures in the next step.

1 1 R Ir[dF(CF3)ppy]2(dtbpy))PF6 R 2 R2 R S S CAN (1.0 eq), P(OEt)3 (3.0 eq) N MeCN (0.1M), 18 h N (EtO)2OP Blue LED(395 nm) CO Me O CO2Me O 2 21a-e 27a-e

F3C

O S S S N N N (EtO)2OP (EtO)2OP (EtO)2OP O CO2Me O CO2Me O CO2Me 27a; 86% 27b; 77% 27c; 75%

S Cl S F3C N N (EtO)2OP (EtO)2OP CO Me O 2 O CO2Me 27d; 61% 27e; 0% Scheme 6. C-6 Phosphonylation of 2-pyridones

In all our previous studies it has been shown that the carboxylic acid is essential for biological activity.1,2 Thus both phosphonate ester and methyl ester were hydrolysed to test their biological activity (Scheme 7). First the phosphonate ester was deprotected using bromo trimethyl silane by heating under MWI at 100 °C for 10 minutes in acetonitrile22. The phosphonic acid was allowed to react further without purification and

9 the methyl ester was hydrolysed with lithium hydroxide by heating under MWI at 65 °C for 30 minutes to give the final compounds 28a-d in 53-72 % yields over two steps. As expected, compound 28a-d had better solubilities compared to the non-phosphonylated analogues. 1 R1 (i) BrSi(CH3)3 R R2 MeCN, MWI,100 °C, R2 S 10 min S N (ii) LiOH (1M, aq) N (HO) OP (EtO)2OP THF:MeOH:H O (3:1:1) 2 2 CO H O CO2Me MWI, 65 °C, 30 min O 2 27a-d 28a-d

F3C

S S N N (HO)2OP (HO)2OP O CO2H O CO2H 28a; 72% 28b; 60%

O S S F3C N N (HO)2OP (HO)2OP O CO2H O CO2H 28c; 58% 28d; 53% Scheme 7. Hydrolysis of phosphonate and methyl esters

Since 2-pyridones with an aryl amide functionality at C-3 have been shown to inhibit the intracellular parasite C. trachomatis,5 we hypothesized that phosphonylation at the C-6 position might improve the solubility while retaining the biological activity. Thus, 2-pyridones 29a-c having an aryl amide at C-3 were phosphonylated under improved reaction conditions (Scheme 8). To our great delight, the reaction conditions proved general as phosphonylated 2-pyridones 30a-c were obtained in good yields after purifying by reverse phase HPLC. Attempts were made to deprotect phosphonate ester using bromotrimethylsilane, however reaction was not successful and only complex mixtures were obtained due to silation at amide.

1 R 1 Ir[dF(CF )ppy] (dtbpy))PF R 2 3 2 6 R 2 S R S CAN (1.0 eq), P(OEt)3 (3.0 eq) N 3 MeCN (0.1M), 18 h N R (EtO)2OP R3 O N Blue LED(395 nm) O H O N 29a-c 30a-c O H

O S S S N N N (EtO)2OP (EtO)2OP (EtO)2OP F O N O N O N O H O H O H 30a; 60% 30b; 65% 30c; 60%

Scheme 8. C-6 Phosphonylation of 2-pyridones

4.4 Plausible Mechanism of the Phosphonylation Reaction

The plausible mechanism of the reaction based on the reported literature methods is depicted in the Scheme 9. The reaction is believed to proceed through the excitation of photocatalyst IrIII by visible light which accepts single electron from the 2-Pyridone 2 to oxidise it to radical cation 3. Ceric ammonium nitrate present in the reaction mixture could take one electron from the reduced IrII to oxidise it back to IrIII to complete the catalytic cycle. The radical cation 3 undergoes reaction with triethyl phosphite 4 to give unstable intermediate 5 which upon deprotonation results in the intermediate 6. In the final step, intermediate 6 releases an ethyl radical17 to furnish the final product 7. The ethyl radical may be quenched by the solvent.

III *Ir IrIII R 1a 1 S oxidant N CeIII

O O SET O SET 2 IrII 1b R reductant CeIV R R S S S N OEt N OEt N P OEt P (EtO)2OP OEt O O O O O CO2Me O OEt O 3 O 7

solvent Et EtH

R R S S H O O N O N O P -H P O O O O O O O O 5 6

Scheme 9. Plausible Mechanism of the Phosphonylation Reaction

4.5 Biological Evaluation

Evaluation of 2-pyridones 28a-c against α-synuclein amyloid formation:

Compound 28a-c were tested for their ability to modulate the formation of α-synuclein amyloid fibrils as measured by a ThT assay. The amyloid formation will lead to an increased fluorescence when ThT binds. For the blank test, wild type α-synuclein is allowed to form amyloid fibers under shaking conditions. The lag time for this process is 10 hours and is detected as an increase in fluorescence signal (Figure 3, A). The bacterial periplasmatic protein CsgC which is a highly effective inhibitor of amyloid formation23, is used as a positive control of amyloid inhibition and no α-synuclein 11 amyloid formation was observed. Compound 2 accelerate the formation of α-synuclein amyloid fibrils with a lag time of 4-5 hours.2b Each experiment was run three times, which is illustrated by three lines (green, red, black) in the diagrams. Unfortunately, C-6 phosphonylation resulted in inactive compounds. All the tested compounds 28a-c did not affect the formation of α-synuclein amyloid formation. Interestingly, compound 28a which is an analogue of the known accelerator 2, lost its activity upon phosphonylation (Figure 3, B and D).

A) B)

18000

18000 16000

16000 14000

14000 12000

12000 10000 CF3 10000

Tht 8000

Tht 8000 6000 S 6000 4000 N 4000 2000 O CO2H 2 2000 0 0 10 20 30 40 50 60 70 0 0 10 20 30 40 50 60 70 Time (hours) Time (hours)

C) D)

18000 18000 16000 16000 14000 14000 12000 12000 CF3 10000 10000

Tht 8000

Tht 8000 6000 S 6000

4000 N 4000 (HO)2OP O CO2H 2000 2000 28a 0 0 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 Time (hours) Time (hours)

E) F)

18000 18000 16000 16000 14000 14000 12000 12000 10000 O 10000

Tht 8000 S

6000 N 6000 N (HO)2OP (HO)2OP 4000 O CO2H 4000 O CO2H 28c 28b 2000 2000

0 0 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 Time (hours) Time (hours)

Figure 3. ThT emission plots recorded at 37°C and during 70 hours after mixing 100 µM of compound together with 70 µM of α-synuclein monomers. Each compound is measured in triplicates (A) Wild type a-synuclein alone. (B) α-synuclein alone in the presence of compound 2 (C) α-synuclein alone in the presence of CsgC. (D-F) α- synuclein alone in the presence of compound 28a, 28b and 28c (compounds did not have any effect on the α -synuclein fibrilization)

Evaluation of 2-pyridones 27d and 30a-c against Chlamydia trachomatis:

The phosphonylated 2-pyridones 28d and 30a-c were tested to inhibit C. trachomatis. The assay consists of infecting a monolayer of HeLa cells with Chlamydia trachomatis 434/ Bu at a MOI of 0.5, for 48h. After that, the inclusions were induced to burst by addition of deionized water and Chlamydia EBs were collected and used to infect fresh HeLa cells. That time, infection proceeded for 44 to 46 h prior fixation of the infected cells by methanol. The cells were stained by the addition of rabbit in-house anti- Chlamydia antibodies, followed by secondary anti-rabbit FITC and DAPI staining of cell nuclei. Analysis of the plates was performed on Array scan automated microscope. In this assay, we calculated the number of Chlamydia inclusions in triplicate samples treated with 2.5 or 1 µM compared to DMSO treated control (as a percentage of growth). In this case, only 30c had a small effect at 2.5 µM, but the effect of the compounds was almost none compared to KSK 2135 treated samples (Figure 4). Unfortunately, all other tested compounds were inactive.

S S S F3C N N (HO) OP N 2 (EtO)2OP (EtO)2OP CO H O 2 O N O N O H 28d 30a 30b O H

O S S

N N (EtO)2OP F O N O N O H O H 30c KSK 213

160

140

120

100

80 2.5 µM 1 µM

% of growth 60

0 27d 30c 30a 30b KSK 213

Figure 4. Evaluation of 2-pyridones 28d and 30a-c against Chlamydia trachomatis

5. Conclusions and Outlook

In conclusion, we have developed a visible-light photoredox catalytic method for the direct C-6 phosphonylation of variedly substituted thiazolino ring fused 2-pyridones utilizing (Ir[dF(CF3)ppy]2(dtbpy))PF6 photocatalyst under very mild reaction conditions at room temperature. The method has the advantage as the 2-pyridone ring could be phosphonylated selectively in the presence of other aryl rings like naphthyl and substituted phenyl. One aim of the study was to make use of this new methodology to potentially improve the physico-chemical properties while retaining the biological activities of ring-fused 2-pyridones. Indeed, we have been successful in improving the solubilities of 2-pyridones. Although, the phosphonylated 2-pyridones were not active in the tested biological systems, the methodology developed herein can be utilized to make libraries of related compounds. It has been seen in the past that 2-pyridones can have different biological targets depending upon their substitution pattern. Thus, phosphonylated 2-pyridones constitute a new subclass that can be tested in various screening campaigns towards a variety of biological targets.

6. Experimental 6.1 General information: Unless stated, all reagents and solvents were used as received from commercial suppliers. All reactions were carried out under an inert atmosphere with dry solvents under anhydrous conditions, unless otherwise indicated. Microwave reactions were performed in sealed vessels using a BiotageÒ Initiator microwave synthesizer; temperatures were monitored by an internal IR probe. TLC was performed on purchased aluminium backed silica gel plates (median pore size 60 Å, fluorescent indicator 254 nm) and detected with UV light at 254 and 366 nm. Automated flash column chromatography was performed using a BiotageÒ Isolera One system and purchased pre-packed silica gel cartridges (BiotageÒ SNAP Cartridge, KP-Sil). Preparative HPLC was performed with a Gilson instrument using a Nucleodur C18 HTec column (25 cm x 21.5 mm; particle size 5 µm). Optical rotation was measured with a Perkin Elmer polarimeter 343 at 25 °C and 589 nm. IR spectra were recorded on a Bruker Alpha-t spectrometer. The samples were prepared as KBr pellets or between NaCl plates, absorbances are given in reciprocal cm. 1H-, 13C- and spectra were recorded on a Bruker Avance III 400 MHz spectrometer with a BBO-F/H SmartprobeTM, a Bruker Avance III HD 600 MHz spectrometer with a CP BBO-H/F, 5 mm cryoprobe, at 298 K, unless other temperature is given. All spectrometers were operated by Topspin 3.5.7. Resonances are given in ppm relative to TMS and calibrated to solvent residual signals (CDCl3: dH = 7.26 ppm; dC = 77.16 ppm. (CD3)2SO: dH = 2.50 ppm; dC = 39.51 ppm. CD3OD dH = 3.31 ppm; dC = 49.00 ppm). The following abbreviations are used to indicate splitting patterns: s = singlet; d = doublet; dd = double doublet; t = triplet; m = multiplet; bs = broad singlet. LC-MS was conducted on a Micromass ZQ mass spectrometer using ES+ ionization unless otherwise stated. HRMS was performed on a mass spectrometer with ESI-TOF (ES+). Amyloid formation was probed by thioflavin T (ThT) fluorescence, with a Fluorostar Omega instrument (BMG Labtech, Germany), using excitation and emission filters of 440 and 480 nm, respectively. Intermediates of general structure 16, 17, 19 and 21 were prepared according to the published procedures.5,6 6.2 General procedure for the synthesis of thiazolines 17a-c: L-cysteine methyl ester hydrochloride (160 mmol 1.4 eq.) was dissolved in DCM (260 mL), cooled down to 0 °C and TEA (172 mmol, 1.5 eq.) was added dropwise. Ethyl acetimidate hydrochloride of general structure 16 (114 mmol, 1.0 eq.) was dissolved in DCM (300 mL) and added dropwise to the reaction mixture. The reaction was stirred over night at room temperature. The reaction mixture was washed with saturated NaHCO3 solution (2 x 80 mL) and brine (1 x 180 mL). The combined aqueous phases were extracted with dichloromethane (2 x 150 mL), the organic layers were combined, dried over anhydrous Na2SO4, filtered and the solvent was removed under reduced pressure. The crude product was purified with automated flash column chromatography (50 g cartridge; 0–100% ethyl acetate in heptane) to thaizolines of general structure 17

CF3

S N

CO2Me (R)-methyl 2-(3-(trifluoromethyl)benzyl)-4,5-dihydrothiazole-4- carboxylate (17a). The compound was prepared following the general procedure. 14 g 15

1 of 16a was converted into 3 g of 17a (19% yield). H NMR (400 MHz, CDCl3): δ 7.52 – 7.36 (m, 4H), 5.10 - 5.06 (m, 1H), 3.92 – 3.84 (m, 2H), 3.76 (s, 3H), 3.58 – 3.43 (m, 2H).

S N

CO2Me (R)-methyl 2-(cyclopropylmethyl)-4,5-dihydrothiazole-4-carboxylate (17b). The compound was prepared following the general procedure. 10 g of 16b was 1 converted into 9 g of 17b (81% yield) H NMR (400 MHz, CDCl3): δ 5.07 (t, J = 9.3 Hz, 1H), 3.81 (s, 3H), 3.62 -3.48 (m, 2H), 2.53 – 2.42 (m, 2H), 1.02 – 0.93 (m, 1H), 0.63 – 0.51 (m, 2H), 0.27 – 0.19 (m, 2H). OMe S N

CO2Me (R)-methyl 2-(methoxymethyl)-4,5-dihydrothiazole-4-carboxylate (17c). The compound was prepared following the general procedure. 6 g of 16c was converted 1 into 4 g of 17c (49% yield) H NMR (400 MHz, CDCl3): δ 5.13-5.07 (m, 1H), 4.33 – 4.25 (m, 2H), 3.79 (s, 3H), 3.58 – 3.46 (m, 2H), 3.41 (s, 3H).

6.3 General procedure for the synthesis of Meldrum’s acid derivative 20a-c:

Meldrum's acid 18 (145 mmol, 1.0 eq.) and the substituted carboxylic acid of general structure 19 (138 mmol, 1.0 eq.) were taken in a dry round bottom flask and flushed with N2 gas. 200 ml of dry DCM was added to the mixture under ice cold condition. DMAP (145 mmol, 1.0eq.) was added to the at 0 °C. After the solution became clear, DCC (1M in DCM, 180 mmol, 1.3 eq.) solution was added dropwise (over the time period of 30 min). The Solution became turbid on full addition of DCC. Completion was confirmed by TLC. Reaction mixture was filtered through the celite pad and washed with 30 mL DCM. The filtrate was collected and washed 5 times with 6% KHSO4. The organic layer was further washed once with brine solution. The organic layer was dried over anhydrous Na2SO4 and evaporated under reduced pressure to yield Meldrum’s acid derivative of general structure 20.

O O

O O 5-(1-hydroxy-2-(naphthalen-1-yl)ethylidene)-2,2-dimethyl-1,3- dioxane-4,6-dione (20a). The compound was prepared following the general procedure. 1 20 g of 19a was converted to 40 g of 20a (93% yield) H NMR (400 MHz, CDCl3): δ 15.48 (s, 1H), 7.98 – 7.96 (m, 1H), 7.89 -7.87 (m, 1H), 7.83 (d, J = 7.7 Hz, 1H), 7.56 – 7.43 (m, 4H), 4.96 (s, 2H), 1.77 (s, 6H).

CF3

O O

O O 5-(1-hydroxy-2-(3-(trifluoromethyl)phenyl)ethylidene)-2,2- dimethyl-1,3-dioxane-4,6-dione (20b). The compound was prepared following the general procedure. 21 g of 19b was converted into 40 g of 20b (88% yield) 1H NMR (400 MHz, CDCl3): δ 15.37 (s, 1H), 7.64 (s, 1H), 7.57 (dd, J = 13.5, 7.8 Hz, 2H), 7.45 (t, J = 7.7 Hz, 1H), 4.48 (s, 2H), 1.73 (s, 6H). Cl HO

O O

O O 5-(2-chloro-1-hydroxyethylidene)-2,2-dimethyl-1,3-dioxane-4,6- dione(20c). The compound was prepared following the general procedure.10 g of 19c 1 was converted into 18 g of 20c (78% yield) H NMR (600 MHz, CDCl3): δ 4.88 (s, 2H), 1.76 (s, 6H). 6.4 General procedure for the synthesis of thiazlino-2-pyridones 21a-e: In a microwave reaction tube equipped with a magnetic stirrer, thiazoline general structure 17 (500mg, 3.1 mmol, 1.0 eq.) and Meldrum’s acid derivative general structure 20 (1.4 g, 6.5 mmol, 2.1 eq.) was dissolved in 1,2-dichloroethane (8 ml). TFA (361 µl, 4.7 mmol, 1.5 eq.) was added, the tube was sealed and heated to 120 °C under microwave irradiations for 3 min. The reaction mixture was cooled to room temperature diluted with DCM (15 ml) and washed with saturated aqueous NaHCO3 (10 ml) followed by brine (10 ml). The aqueous phases were re-extracted with DCM (15 ml each). The organic phases were combined, dried, filtered and evaporated. The compound was purified with automated flash column chromatography (50 g cartridge; 0–100% ethyl acetate in heptane) to yield 2-pyridones of general structure 21a-e. CF3

S N O CO2Me (R)-methyl 7-(naphthalen-1-ylmethyl)-5-oxo-8-(3- (trifluoromethyl)phenyl)-3,5-dihydro-2H-thiazolo[3,2-a]pyridine-3-carboxylate (21a). The compound was prepared following the general procedure. 2.5g of 17a was 1 converted into 3.19g of 21a (76% yield). H NMR (400 MHz, CDCl3): δ 7.83 – 7.81 (m, 1H), 7.74 (d, J = 8.2 Hz, 1H), 7.59 – 7.54 (m, 3H), 7.49 – 7.34 (m, 5H), 7.18 (d, J = 6.8 Hz, 1H), 5.93 (t, J = 6.5 Hz, 1H), 5.64 (dd, J = 8.5, 2.3 Hz, 1H), 4.02 – 3.85 (m, 2H), 3.84 (s, 3H), 3.69 (t, J = 10.2 Hz, 1H), 3.53 – 3.44 (m, 1H).

S N O CO2Me (R)-methyl 8-cyclopropyl-7-(naphthalen-1-ylmethyl)-5-oxo- 3,5-dihydro-2H-thiazolo[3,2-a]pyridine-3-carboxylate (21b). The compound was prepared following the general procedure. 1.4 g of 17b was converted into 750 mg of 1 21b (76% yield) H NMR (600 MHz, CDCl3): δ 7.87 (dd, J = 6.1, 3.3 Hz, 1H), 7.79 (dd, J = 11.0, 5.9 Hz, 2H), 7.49 – 7.44 (m, 2H), 7.43 – 7.39 (m, 1H), 7.28 – 7.26 (m, 1H), 5.75 (s, 1H), 5.56 (dd, J = 8.5, 2.2 Hz, 1H), 4.50 (d, J = 17.2 Hz, 1H), 4.35 (d, J = 17.2 17

Hz, 1H), 3.78 (s, 3H), 3.66 (dd, J = 11.7, 8.6 Hz, 1H), 3.50 (dd, J = 11.8, 2.1 Hz, 1H), 1.67–1.62 (m, 1H), 1.00 – 0.87 (m, 2H), 0.79 – 0.69 (m, 2H). O S N O CO2Me (R)-methyl 8-methoxy-7-(naphthalen-1-ylmethyl)-5-oxo- 3,5-dihydro-2H-thiazolo[3,2-a]pyridine-3-carboxylate (21c). The compound was prepared following the general procedure. 500 mg of 17c was converted into 800 mg of 1 21c (80% yield) H NMR (400 MHz, CDCl3): δ 7.88 – 7.81 (m, 2H), 7.79 (d, J = 8.2 Hz, 1H), 7.50 – 7.44 (m, 2H), 7.43 – 7.41 (m, 1H), 7.33 (d, J = 6.5 Hz, 1H), 5.80 (s, 1H), 5.55 (dd, J = 8.4, 2.2 Hz, 1H), 4.29 (dd, J = 49.6, 16.8 Hz, 2H), 3.80 (s, 3H), 3.77 (d J = 11.7, 8.4 Hz, 1H) 3.74 (s, 3H), 3.60 (dd, J = 11.7, 2.2 Hz, 1H).

F3C S N

O CO2Me (R)-methyl 8-cyclopropyl-5-oxo-7-(3- (trifluoromethyl)benzyl)-3,5-dihydro-2H-thiazolo[3,2-a]pyridine-3-carboxylate (21d). The compound was prepared following the general procedure. 3.7 g of 17d was 1 converted into 2.1 g of 21d (70% yield). H NMR (400 MHz, CDCl3): δ 7.50 (d, J = 7.7 Hz, 1H), 7.45 – 7.41 (m, 2H), 7.37 (d, J = 7.7 Hz, 1H), 6.00 (s, 1H), 5.60 (dd, J = 8.5, 2.2 Hz, 1H), 4.11 – 3.97 (m, 2H), 3.80 (s, 3H), 3.66 (dd, J = 11.7, 8.6 Hz, 1H), 3.50 (dd, J = 11.7, 2.2 Hz, 1H), 1.41 – 1.34 (m, 1H), 0.93 – 0.86 (m, 2H), 0.69 – 0.59 (m, 2H).

Cl S N O CO2Me (R)-methyl 7-(chloromethyl)-8-cyclopropyl-5-oxo-3,5-dihydro- 2H-thiazolo[3,2-a]pyridine-3-carboxylate (21e). The compound was prepared following the general procedure. 5.5 g of 17b was converted into 6.48 g of 21e (78% 1 yield). H NMR (600 MHz, CDCl3): δ 6.37 (s, 1H), 5.56 (dd, J = 8.6, 1.9 Hz, 1H), 4.60 (d, J = 12.5 Hz, 1H), 4.49 (d, J = 12.5 Hz, 1H), 3.76 (s, 3H), 3.65 (dd, J = 11.7, 8.7 Hz, 1H), 3.48 (dd, J = 11.7, 2.1 Hz, 1H), 1.67–1.63 (m, 1H), 0.94– 0.93 (m, 2H), 0.64–0.61 (m, 2H).

6.5 General procedure for the synthesis of amides 29a-b5:

NH2 R1 S R S R N HATU, DIPEA, DMF N rt, 18 h O OH O N O O H 4 29a-b The carboxylic acid (0.79 mmol, 1.0 eq.) was dissolved in anhydrous DMF (15 mL), amine (1.19 mmol, 1.5 eq.), DIPEA (1.19 mmol, 1.5eq.), and HATU (0.87 mmol, 1.1 eq.) added, and the reaction stirred at room temperature for18 h. The reaction mixture was diluted with DCM (30 mL), washed with aqueous HCl (1M), and brine. The organic layer was dried over anhydrous Na2SO4 and was evaporated under reduced pressure. The crude product was purified by automated column chromatography (25 g cartridge; eluting with Heptane/EtOAc, 0-100%)

S N

O N O H 8-cyclopropyl-7-(naphthalen-1-ylmethyl)-5-oxo-N- phenyl-3,5-dihydro-2H-thiazolo[3,2-a]pyridine-3-carboxamide (29a). The compound was prepared following the general procedure. 300 mg of 4 was converted 1 into 294 mg of 29a (81% yield). H NMR (400 MHz, (CD3)2SO): δ 10.37 (s, 1H), 7.98 – 7.95 (m, 1H), 7.90 – 7.87 (m, 2H), 7.56 – 7.51 (m, 5H), 7.38 (d, J = 6.6 Hz, 1H), 7.31 (d, J = 7.7 Hz, 2H), 7.05 (t, J = 7.4 Hz, 1H), 5.47 (dd, J = 9.1, 2.0 Hz, 1H), 4.46 (dd, J = 37.1, 17.3 Hz, 2H), 3.87 (dd, J = 11.9, 9.2 Hz, 1H), 3.53 (dd, J = 11.9, 2.1 Hz, 1H), 1.76 – 1.69 (m, 1H), 0.96 – 0.89 (m, 2H), 0.78 – 0.76 (m, 1H), 0.68 – 0.65 (m, 1H).

S N

O N O H 8-cyclopropyl-7-(naphthalen-1-ylmethyl)-5-oxo-N-(p- tolyl)-3,5-dihydro-2H-thiazolo[3,2-a]pyridine-3-carboxamide (29b) The compound was prepared following the general procedure. 400 mg of 4 was converted into 350mg 1 of 29 b (71% yield). H NMR (400 MHz, CDCl3): δ 10.28 (d, J = 22.5 Hz, 1H), 7.89 – 7.86 (m, 1H), 7.80 – 7.73 (m, 2H), 7.48 – 7.40 (m, 5H), 7.24 (s, 1H), 7.05 (d, J = 8.3 Hz, 2H), 5.86 – 5.81 (m, 2H), 4.43 (dd, J = 45.5, 17.4 Hz, 2H), 4.16 – 4.09 (m, 1H), 3.66 – 3.56 (m, 1H), 2.27 (s, 3H), 1.71 – 1.65 (m, 1H), 1.06 – 0.99 (m, 1H), 0.95 – 0.88 (m, 1H), 0.82 – 0.76 (m, 1H), 0.72 – 0.66 (m, 1H).

6.6 General Procedure for Photoredox catalyzed phosphonylation of 2- pyridones: To the 2-pyridone of general structure 21 or 29 (0.4 mmol, 1eq) was added

CAN (0.4 mmol, 1eq) and (Ir[dF(CF3)ppy]2(dtbpy))PF6 (0.008 mmol, 0.02eq), followed by the addition of MeCN (3.9 ml, 0.1M). Nitrogen was passed through the mixture for about five minutes. Under nitrogen, triethyl phosphite (1.2 mmol, 3 eq.) was added. Reaction mixture was kept under nitrogen for 10 more minutes. The reaction mixture was then placed for irradiation under 395 nm LED. The completion was confirmed by LCMS. The reaction mixture was transferred to a separation funnel and diluted with water (10ml) and EtOAc (50ml).The organic layer was washed brine three times

(3x20ml). The organic layers were combined and dried over Na2SO4, filtered and evaporated under vacuum to afford crude product. The crude product was purified by automated flash chromatography, first eluted with (Heptne / EtOAc, 0-100%), then with DCM/Methanol (0-20%). The desired product eluted in 5% DCM/Methanol.Two eluents were used to remove non-polar impurities.

F3C

S N (EtO)2OP O CO2Me 6-(Diethoxy-phosphoryl)-7-naphthalen-2-ylmethyl-5-oxo-8- (3-trifluoromethyl-phenyl)-2,3-dihydro-5H-thiazolo[3,2-a]pyridine-3-carboxylic acid methyl ester (27a): Compound was prepared following the general procedure. The reaction was completed after 18h. The crude product was purified with preparative HPLC (H2O/MeCN + 0.75% HCOOH; 30–100% in 35 min., 100% for 10 min.) The fraction containing the desired product was evaporated to remove the organic solvent. 19

Remaining aqueous part was extracted with dichloromethane (20 ml). The organic layer was dried under reduced pressure to give pure product. 198 mg of 21a was converted into 219mg of 27a (86% yield). A mixture of atropoisomers. IR (KBr cm-1): ν 2984, 1 1747, 1649, 1473, 1326, 1029, 970. H NMR (400 MHz, CDCl3) δ 7.72 (d, J = 7.9 Hz, 1H), 7.63 (d, J = 8.2 Hz, 1H), 7.55 (dd, J = 8.4, 2.9 Hz, 1H), 7.37 – 7.26 (m, 3H), 7.23 – 6.91 (m, 5H), 5.76 (dd, J = 8.8, 2.6 Hz, 1H), 4.91 – 4.65 (m, 2H), 4.19 – 4.01 (m, 4H), 3.87 (a pair of singlets, 3H), 3.72 – 3.65 (m, 1H), 3.47 – 3.43 (m, 1H), 1.25 – 1.18 (m, 13 6H). C NMR (100 MHz, CDCl3) δ 168.1, 162.5, 162.4, 160.1, 153.1, 136.5, 135.4, 133.5, 133.2, 131.5, 129.0, 128.9, 128.5, 128.4, 127.0, 126.9, 125.8, 125.5, 125.3, 125.0, 122.8, 122.7, 116.4, 116.2, 113.2, 64.48, 62.5, 62.4, 53.6, 53.5, 33.7, 31.5, 16.5, 16.4, + + 16.3. (ESI-TOF) m/z [M+H] calcd for C31H29F3NO6PS 632.1484, found 632.1478.

S N (EtO)2OP O CO2Me 8-Cyclopropyl-6-(diethoxy-phosphoryl)-7-naphthalen-2- ylmethyl-5-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyridine-3-carboxylic acid methyl ester (27b). Compound was prepared following the general procedure. The reaction was completed after 18h. The crude product was purified with automated flash column chromatography (25 g cartridge; Heptane/EtOAc 0-100% then DCM/Methnol 0-5%) 156 mg of 21b was converted to162 mg of 27b (77% yield). IR (KBr cm-1): ν 2981, 1 1753, 1644, 1471, 1214, 1028, 961. H NMR (400 MHz, CDCl3): δ 8.20 (d, J = 8.4 Hz, 1H), 7.85 (d, J = 7.9 Hz, 1H), 7.68 (d, J = 8.3 Hz, 1H), 7.58 – 7.48 (m, 2H), 7.30 (t, J = 7.7 Hz, 1H), 6.82 (d, J = 7.1 Hz, 1H), 5.74 (d, J = 7.3 Hz, 1H), 5.27 (dd, J = 39.5, 15.4 Hz, 2H), 4.07 – 3.93 (m, 4H), 3.83 (s, 3H), 3.75 – 3.66 (t, J = 2.4 Hz, 1H), 3.51 (dd, J = 11.8, 2.2 Hz, 1H), 1.22 – 1.18 (m, 1H), 1.10 (t, J = 7.0 Hz, 6H), 0.71 – 0.58 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 168.1, 164.6, 164.5, 159.9, 159.8, 153.5, 153.5, 135.8, 133.4, 131.8, 128.4, 126.3, 125.8, 125.4, 125.1, 123.6, 123.1, 114.8, 114.6, 114.4, 112.5, 63.4, 63.3, 62.0, 61.9, 61.7, 61.6, 61.5, 53.0, 33.0, 31.0, 11.5, 7.9, 7.4. HRMS (ESI-TOF) m/z + + [M + H] calcd for C27H30NO6PS 528.1610, found 528.1610.

O S N (EtO)2OP O CO2Me 6-(Diethoxy-phosphoryl)-8-methoxy-7-naphthalen-2- ylmethyl-5-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyridine-3-carboxylic acid methyl ester (27c). The compound was prepared following the general procedure. The reaction was completed after 18h. The crude product was purified with automated flash column chromatography (25 g cartridge; Heptane/EtOAc 0-100% then DCM/Methnol 0- 5%).150 mg of 21c was converted into 152 mg 27c (75% yield). IR (KBr cm-1): ν 2982, 1 1754, 1645, 1492, 1214, 1020, 982. H NMR (400 MHz, CDCl3): δ 8.18 (d, J = 8.4 Hz, 1H), 7.83 (d, J = 7.6 Hz, 1H), 7.67 (d, J = 8.2 Hz, 1H), 7.58 – 7.45 (m, 2H), 7.32 – 7.28 (m, 1H), 6.95 (d, J = 6.6 Hz, 1H), 5.67 (dd, J = 8.7, 2.5 Hz, 1H), 5.06 – 4.95 (m, 2H), 4.17 (m, 4H), 3.83 (s, 3H), 3.77 (dd, J = 11.8, 8.7 Hz, 1H), 3.57 (dd, J = 11.8, 2.5 Hz, 13 1H), 3.30 (s, 3H), 1.16 – 1.08 (m, 6H). C NMR (100 MHz, CDCl3) δ 167.7, 159.2, 159.1, 159.0, 158.9, 144.9, 137.2, 137.0, 135.6, 133.4, 131.7, 128.4, 126.5, 125.9, 125.4, 124.9, 123.9, 123.3, 114.0, 112.1, 64.3, 64.2, 63.6, 62.1, 62.0, 61.5, 61.4, 60.6, 53.2, + + 31.8, 30.1. HRMS (ESI-TOF) m/z [M + H] calcd for C25H28NO7PS 518.1402, found 518.1406.

S F3C N (EtO)2OP O CO2Me 8-Cyclopropyl-6-(diethoxy-phosphoryl)-5-oxo-7-(3- trifluoromethyl-benzyl)-2,3-dihydro-5H-thiazolo[3,2-a] pyridine-3-carboxylic acid methyl ester (21d). The compound was prepared following the general procedure. The reaction was completed after 18h. The crude product was purified with automated flash column chromatography (25 g cartridge; Heptane/EtOAc 0-100% then DCM/Methnol 0-5%). 163 mg of 21d was converted to 133 mg of 27d (61%). IR (KBr cm-1): ν 2983, 1 1754, 1645, 1471, 1330, 1030, 964. H NMR (600 MHz, CDCl3): δ 7.45 – 7.34 (m, 4H), 5.68 (dd, J = 8.8, 2.0 Hz, 1H), 5.16 (d, J = 13.1 Hz, 1H), 4.75 (d, J = 13.4 Hz, 1H), 3.78 (s, 3H), 3.67 (dd, J = 11.7, 9.0 Hz, 1H), 3.47 (dd, J = 11.8, 2.3 Hz, 1H), 1.31 (m, 1H), 1.23 (m, 6H), 1.18-1.13 (m, 1H), 0.95 – 0.87 (m, 1H), 0.86 – 0.79 (m, 1H), 0.67 – 0.57 13 (m, 1H). C NMR (100 MHz, CDCl3) δ 168.3, 164.0, 160.0, 154.3, 140.8, 132.0, 130.8, 130.5, 128.8, 125.7, 125.0, 125.0, 123.0, 115.0, 114.8, 114.0, 112.1, 63.7, 62.6, 62.5, 62.4, 62.3, 53.4, 36.8, 36.0, 31.4, 17.5, 17.4, 16.5, 16.4, 16.3, 12.1, 9.0, 8.5. HRMS (ESI- + + TOF) m/z [M + H] calcd for C24H27F3NO6PS 546.1327 Found 546.1360.

S N (EtO)2OP O N O H 8-Cyclopropyl-7-naphthalen-2-ylmethyl-5-oxo-3- phenylcarbamoyl-2,3-dihydro-5H-thiazolo[3,2-a]pyridin-6-yl)-phosphonic acid diethyl ester (30a). The compound was prepared following the general procedure. The reaction was monitored by LCMS. The reaction was not completed after 18 h so 2 equivalents of triethyle phosphite was added and kept stirring for additional 6 hours. The crude product was purified with automated flash column chromatography (25 g cartridge; heptane/ethylacetate from 0 to 100%) and then (DCM/MeOH step gradient 0 to 10% very slowly). Product eluted at DCM/MeOH 5%. Different fractions were checked with TLC, combined and concentrated. The 1HNMR showed some triethyl phosphite left in product so reverse phase HPLC was done for purification. The compound was re-dissolved in DMSO (1 ml) and purified with preparative HPLC (H2O/MeCN + 0.75% HCOOH; 30–100% in 35 min., 100% for 10 min.) The fractions containing the desired product was diluted with H2O (1:1) and freeze-dried. 65mg of 29a was converted to 55mg of 30a (65%yield). IR (KBr, cm-1): ν 3432, 1701, 1640, 1 1471, 1027, 961. H NMR (400 MHz, CDCl3): δ 10.48 (s, 1H), 8.18 (d, J = 8.4 Hz, 1H), 7.86 (d, J = 7.3 Hz, 1H), 7.70 (d, J = 8.3 Hz, 1H), 7.62 – 7.49 (m, 4H), 7.32 – 7.27 (m, 3H), 7.11 (t, J = 7.4 Hz, 1H), 6.76 (d, J = 7.0 Hz, 1H), 6.16 (d, J = 8.1 Hz, 1H), 5.37 (d, J = 15.3 Hz, 1H), 5.10 (d, J = 15.3 Hz, 1H), 4.19 (d, J = 11.5 Hz, 1H), 4.13 – 3.93 (m, 4H), 3.77 (dd, J = 11.2, 8.6 Hz, 1H), 1.29-1.23 (m,1H), 1.14-1.10 (m, 6H), 0.75 – 0.70 13 (m, 1H), 0.67 (dd, J = 14.8, 7.2 Hz, 2H), 0.54-0.50 (m, 1H). C NMR (151 MHz, CDCl3) δ 164.9, 164.8, 164.4, 162.0, 162.0, 138.1, 135.6, 133.8, 132.1, 129.1, 128.8, 126.9, 126.4, 125.9, 125.4, 124.6, 123.9, 123.3, 120.1, 65.9, 62.6, 62.5, 33.5, 30.0, 16.4, 16.3, + + 12.2, 8.8, 7.2. HRMS (ESI-TOF) m/z [M + H] calcd for C32H33N2O5PS 589.1926 found 589.1927.

S N (EtO)2OP O N O H (8-Cyclopropyl-7-naphthalen-2-ylmethyl-5-oxo-3-p- tolylcarbamoyl-2,3-dihydro-5H-thiazolo[3,2-a]pyridin-6-yl)-phosphonic acid

21 diethyl ester (30b). The compound was was prepared following the general procedure. The reaction was monitored by LCMS. The reaction was not complete after 18 hours so two more equivalents of triethyle phosphite were added and was kept stirring for additional 6 hours. The crude product was purified with automated flash column chromatography (25 g cartridge using heptane/ethylacetate from 0 to 100%) and then with DCM/MeOH step gradient 0 to 10% very slowly. Different fractions were checked with TLC, combined and concentrated. The 1HNMR showed some triethyl phosphite left in the product so HPLC was used for purification. The compound was re-dissolved in DMSO (1 ml) and purified with preparative HPLC (H2O/MeCN + 0.75% HCOOH; 30–100% in 35 min., 100% for 10 min.) The fraction containing the desired product was diluted with H2O (1:1) and freeze-dried. 186 mg of 29b was converted to 121 mg of 30b -1 1 (50%). IR (KBr cm ): ν 2981, 1700,1642, 1471, 1027, 961. H NMR (600 MHz, CDCl3): δ 10.40 (s, 1H), 8.18 (d, J = 8.4 Hz, 1H), 7.86 (d, J = 8.0 Hz, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.58 (dd, J = 11.1, 4.1 Hz, 1H), 7.53 – 7.49 (m, 3H), 7.29 (t, J = 7.7 Hz, 1H), 7.11 (d, J = 8.2 Hz, 2H), 6.75 (d, J = 7.0 Hz, 1H), 6.17 (d, J = 7.5 Hz, 1H), 5.37 (d, J = 15.0 Hz, 1H), 5.10 (d, J = 15.3 Hz, 1H), 4.19 (d, J = 11.4 Hz, 1H), 4.11 – 3.92 (m, 4H), 3.80 – 3.67 (m, 1H), 2.31 (s, 3H), 1.25 (dd, J = 13.9, 6.9 Hz, 1H), 1.12 (dd, J = 12.9, 6.3 Hz, 6H), 0.77 – 0.70 (m, 1H), 0.67 (dd, J = 14.8, 7.2 Hz, 2H), 0.52 (m,1H). 13C NMR (151 MHz, CDCl3): δ 165.0, 164.9, 164.1, 162.0, 161.9, 135.5, 135.4 134.3, 133.8, 132.0, 129.5, 128.9, 127.0, 126.4, 126.0, 125.4, 123.8, 123.3, 120.1, 66.1, 62.7, 62.7, 33.5, 30.3, 21.3, 16.4, 16.3, 12.2, 8.7, 7.2. HRMS (ESI-TOF) m/z [M + H]+ calcd for + C33H35N2O5PS 603.2083 found 603.2087. O S N (EtO)2OP F O N O H [7-(2,3-Dimethyl-benzyl)-3-(3-fluoro-5-methyl- phenylcarbamoyl)-8-methoxy-5-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyridin-6-yl]- phosphonic acid diethyl ester (30c). The compound was prepared following the general procedure. The reaction was monitored by LCMS. The reaction was not complete after 18 hours so 2 more equivalents of triethyle phosphite was added. The reaction was finished after 24hrs. The crude product was purified with automated flash column chromatography (25 g cartridge using heptane/ethylacetate from 0 to 100% and then DCM/MeOH step gradient 0 to 10% very slowly). Different fractions were checked with TLC, combined and concentrated. 1HNMR showed some triethyl phosphite left in the product so HPLC was done for purification. The compound was re-dissolved in DMSO (1 ml) and purified with preparative HPLC (H2O/MeCN + 0.75% HCOOH; 30– 100% in 35 min., 100% for 10 min.) The fraction containing the desired product was diluted with H2O (1:1) and freeze-dried.100mg of 29c was converted to 79 mg of 30c (61%). IR (KBr cm-1): ν 3425, 1706, 1628, 1482, 1024, 960.1H NMR (600 MHz, CDCl3): δ 10.64 (s, 1H), 7.36 (d, J = 10.5 Hz, 1H), 7.10 (s, 1H), 6.98 (d, J = 7.4 Hz, 1H), 6.92 (t, J = 7.6 Hz, 1H), 6.63 (d, J = 9.2 Hz, 1H), 6.52 (d, J = 7.6 Hz, 1H), 6.12 (d, J = 6.8 Hz, 1H), 4.47 (dd, J = 36.0, 14.6 Hz, 2H), 4.24 (d, J = 11.4 Hz, 1H), 4.10 – 4.06 (m, 4H), 3.83 (t, J = 9.7 Hz, 1H), 3.39 (s, 3H), 2.32 – 2.29 (m, 9H), 1.24-1.18 (m, 6H). 13 C NMR (151 MHz, CDCl3) δ 164.2, 163.8, 162.2, 160.9, 160.3, 160.2, 140.8, 139.1, 139.0, 137.3, 136.7, 134.7, 128.2, 125.3, 124.7, 116.2, 112.2, 112.1, 104.9, 104.7, 66.5, 63.0, 62.6, 61.0, 31.6, 30.8, 21.5, 21.0, 16.5, 16.4, 16.3, 15.4. HRMS (ESI-TOF) m/z [M + + + H] calcd for C29H34FN2O6PS 589.1937 found 589.1938.

6.7 General procedure for the hydrolysis of phosphonylated 2- pyridones: The phosphonylated 2-pyridones 27a-d were hydrolysed and the yields were noted over two steps. In the first step, the phosphonylated product (1eq) was taken 22 into dried 5 ml microwave vial, anhydrous MeCN (1 mL) was added followed by the addition of bromotrimethyl silane (4 eq.). The mixture was heated under MWI at 100 °C for 10 minutes. The reaction was monitored with LCMS. After completion, the reaction mixture was diluted with H2O and MeCN was evaporated under vacuum. The remaining aqueous part was treated with HCl (6M), diluted with MeOH and extracted with EtOAc.

The organic layer was dried over Na2SO4 and concentrated under vacuum to afford the curde phosphonic acid as a whitish semisolid. The Crude phosphonic acid was used as such in next step without further purification. Crude phosphonic acid product (1eq.) and aq. LiOH (1M, 6 eq.) were taken in a microwave vial and added THF/MeOH/H2O 3:1:1 (0.07 M). the reaction mixture was heated under MWI for 30 min at 65 °C. LC-MS showed full conversion to the desired product. The volatile solvents were evaporated, and the aqueous layer was acidified with HCl (6 M) to pH < 3. The precipitates formed was filtered and washed with water to afford the acid. The compound was re-dissolved in DMSO (1 ml) and purified with preparative HPLC (H2O/MeCN + 0.75% HCOOH; 30–100% in 40 min., 100% for 10 min.) The fraction containing the desired product was diluted with H2O (1:1) and freeze-dried.

F3C

S N (HO)2OP O CO2H 7-Naphthalen-2-ylmethyl-5-oxo-6-phosphono-8-(3- trifluoromethyl-phenyl)-2,3-dihydro-5H-thiazolo[3,2-a]pyridine-3-carboxylic acid (28a). The compound was prepared following the general procedure. 100mg of 27a was ° -1 converted to 91mg of 28a (Yield 52%). [α]D -2.8 (c 0.20, DMSO); IR (KBr cm ): ν 1 3434, 1624, 1493, 1328, 1167, 1127. H NMR (600 MHz, (CD3)2SO): δ 7.77 (d, J = 8.1 Hz, 1H), 7.66 (d, J = 8.2 Hz, 1H), 7.60 (d, J = 8.5 Hz, 1H), 7.37 (t, J = 7.4 Hz, 1H), 7.34 – 7.25 (m, 4H), 7.19 (d, J = 4.7 Hz, 1H), 7.00 (dd, J = 13.2, 7.1 Hz, 1H), 6.91 (d, J = 24.2 Hz, 1H), 5.73 (d, J = 8.1 Hz, 1H), 4.96-4.81 (m, 1H), 4.66-4.49 (m, 1H), 3.92 (dd, 13 J = 20.9, 11.5 Hz, 1H), 3.59 – 3.56 (m, 1H). C NMR (151 MHz, (CD3)2SO): δ 168.7, 162.2, 162.1, 159.5, 153.3, 136.2, 136.1, 134.5, 134.4, 133.8, 132.8, 130.8, 129.4, 128.2, 126.4, 126.0, 125.9, 125.6, 125.3, 125.2, 125.0, 125.0, 65.0, 33.4, 31.9, 31.8. HRMS + + (ESI-TOF) m/z [M + H] calcd for C26H19F3NO6PS 562.0701 found 562.0718.

S N (HO)2OP O CO2H 8-Cyclopropyl-7-naphthalen-2-ylmethyl-5-oxo-6- phosphono-2,3-dihydro-5H-thiazolo[3,2-a]pyridine-3-carboxylic acid (28b). The compound was prepared following the general procedure. 192mg of 27b was converted ° -1 to 100mg of 28b (Yield 60%). [α]D -15.6 (c 0.20, DMSO); IR (KBr cm ): ν 3430, 1735, 1 1621, 1486, 1161, 1148, 990, 947. H NMR (600 MHz, (CD3)2SO): δ 8.28 (d, J = 8.5 Hz, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.76 (d, J = 8.2 Hz, 1H), 7.63 – 7.60 (m, 1H), 7.55 (t, J = 7.4 Hz, 1H), 7.36 (t, J = 7.7 Hz, 1H), 6.81 (d, J = 7.1 Hz, 1H), 5.71 (dd, J = 9.3, 1.5 Hz, 1H), 5.17 (d, J = 15.8 Hz, 1H), 5.06 (d, J = 15.8 Hz, 1H), 3.93 (dd, J = 11.9, 9.4 Hz, 1H), 3.66 (dd, J = 12.0, 1.4 Hz, 1H), 0.95 – 0.93 (m, 1H), 0.58-0.57 (m, 4H). 13C NMR (151 MHz, (CD3)2SO): δ 168.9, 162.2, 162.1, 153.7, 134.8, 133.2, 131.5, 128.6, 126.3,

126.1, 125.7, 125.5, 124.3, 123.4, 116.6, 116.5, 116.1, 115.0, 64.1, 33.5, 31.4, 11.4, 7.9, + + 7.0. HRMS (ESI-TOF) m/z [M + H] calcd for C22H20NO6PS 458.0827 found 458.0849.

O S N (HO)2OP O CO2H 8-Methoxy-7-naphthalen-2-ylmethyl-5-oxo-6-phosphono-2,3- dihydro-5H-thiazolo[3,2-a]pyridine-3-carboxylic acid (28c). The compound was prepared following the general procedure.100mg 27c was converted into 52 mg of 28c ° -1 (yield 58 %). [α]D –0.8 (c 0.20, DMSO); IR (KBr cm ): ν 3433, 1753, 1615, 1508, 1021, 955, 796. 1H NMR (600 MHz, DMSO) δ 8.28 (d, J = 8.4 Hz, 1H), 7.95 (d, J = 7.9 Hz, 1H), 7.77 (d, J = 8.2 Hz, 1H), 7.61 (dd, J = 11.2, 4.1 Hz, 1H), 7.56 (t, J = 7.4 Hz, 1H), 7.37 (t, J = 7.7 Hz, 1H), 6.97 (d, J = 7.0 Hz, 1H), 5.69 – 5.60 (m, 1H), 4.86 (dd, J = 37.2, 15.3 Hz, 2H), 4.04 (dd, J = 11.8, 9.3 Hz, 1H), 3.76 (d, J = 11.8 Hz, 1H), 3.27 (s, 3H). 13C NMR (151 MHz, DMSO) δ 168.6, 160.9, 160.8, 156.1, 145.7, 138.2, 138.1, 135.0, 133.2, 131.4, 128.5, 126.4, 126.1, 125.7, 125.4, 124.8, 123.6, 115.5, 114.4, 64.4, 59.9, + + 32.4, 30.7. HRMS (ESI-TOF) m/z [M + H] calcd for C20H18NO7PS 448.0620 found 448.0639.

S F3C N (HO)2OP O CO2H 8-Cyclopropyl-5-oxo-6-phosphono-7-(3-trifluoromethyl- benzyl)-2,3-dihydro-5H-thiazolo[3,2-a]pyridine-3-carboxylic acid (28d). The compound was prepared following the general procedure.100mg of 27d was converted ° -1 to 45mg of 28d (yield 55%). [α]D –15.2 (c 0.20, DMSO); IR (KBr cm ): ν 3290, 1 1741,1637, 1484, 1333, 1122, 789. H NMR (600 MHz, (CD3)2SO): δ 7.65 (s, 1H), 7.55– 7.54 (m, 1H), 7.50 (d, J = 5.0 Hz, 2H), 5.64 (dd, J = 9.3, 1.4 Hz, 1H), 4.90 (d, J = 15.1 Hz, 1H), 4.58 (d, J = 15.1 Hz, 1H), 3.89-3.86 (m, 1H), 3.62 (dd, J = 11.9, 1.3 Hz, 1H), 0.93– 0.91 (m, 2H), 0.83-0.82 (m,1 H), 0.64-0.62 (m,1H), 0.54 – 0.49 (m, 1H). 13C NMR (151 MHz, (CD3)2SO): δ 168.9, 162.3, 162.2, 161.1, 161.0, 154.4, 139.9, 132.8, 129.3, 129.2, 128.9, 127.1, 125.3, 123.5, 123.0, 121.7, 116.4, 116.3, 115.8, 114.7, 64.4, + + 36.7, 31.6, 11.8, 8.8, 8.2. HRMS (ESI-TOF) m/z [M + H] calcd for C19H17F3NO6PS 476.0545 found 476.0556.

Acknowledgement Firstly, I would like to express my special thanks to my supervisor Prof. Fredrik Almqvist for accepting me as a master`s degree student and to give me a great chance to work on this project to improve my organic chemistry synthesis skills, for his valuable guidance and support. I would also thank my Lab supervisor Micheal Saleeb for providing me indispensable advice, support, cooperation and sharing his knowledge by answering me time to time. My sincere thanks go to Pardeep Singh for his guidance and valuable suggestions in the laboratory work. I would extend my gratitude to Souvik Sarkar for explain me how to do column chromatography and HPLC. I would also like to thank Jaideep Bharate for his support during lab work and describing to me how to do IR spectroscopy, Mohit Tyagi for explaining to me how to run Polarimeter and Marcus Carlsson for HRMS analysis. I would also thank to Anders, Anita and Ingeborg for their guidance. I would also extend my thank to Carlos Nunez Otero and Jörgen for testing biological activity of compounds. Finally, I would thank to my Family for cooperating me and helping me all the times.

References 1. a) V. Åberg, F. Almqvist, Org. Biomol. Chem. 2007, 5, 1827–1834. b) J. S. Pinkner, H. Remaut, F. Buelens, E. Miller, V. Åberg, N. Pemberton, M. Hedenström, A. Larsson, P. Seed, G. Waksman, S. J. Hultgren, F. Almqvist, Proc. Natl. Acad. Sci. USA 2006, 103 (47), 17897–17902. c) E. Chorell, J. S. Pinkner, G. Phan, S. Edvinsson, F. Buelens, H. Remaut, G. Waksman, S. J. Hultgren, F. Almqvist, J. Med. Chem. 2010, 53, 5690–5695. 2. a) V. Åberg, F. Norman, E. Chorell, A. Westermark, A. Olofsson, A. E. Sauer- Eriksson, F. Almqvist, Org. Biomol. Chem. 2005, 3, 2817–2823. b) I. Horvath, C. F. Weise, E. K. Andersson, E. Chorell, M. Sellstedt, C. Bengtsson, A. Olofsson, S. J. Hultgren, M. R. Chapman, M. Wolf-Watz, F. Almqvist, P. Wittung-Stafshede, J. Am. Chem. Soc. 2012, 134 (7), 3439–3444. c) I. Horvath, M. Sellstedt, C. Weise, L-M. Nordvall, G. K. Prasad, A. Olofsson, G. Larsson, F. Almqvist, P. Wittung-Stafshede, Arch. Biochem. Biophy. 2013, 532 (2), 84– 90. 3. a) J. A. D. Good, C. Andersson, S. Hansen, J. Wall, K. S. Krishnan, A. Begum, C. Grundstrom, M. S. Niemiec, K. Vaitkevicius, E. Chorell, P. Wittung- Stafshede, U. H. Sauer, A. E. Sauer-Eriksson, F. Almqvist, J. Johansson, Cell Chem Biol 2016, 23, 404–414. b) M. Kulén, M. Lindgren, S. Hansen, A. G. Cairns, C. Grundström, A. Begum, I. van der Lingen, K. Brännström, M. Hall, U. H. Sauer, J. Johansson, A. E. Sauer-Eriksson, F. Almqvist, J. Med. Chem. 2018, 61 (9), 4165–4175. 4. K. Flentie, G. A. Harrison, H. Tükenmez, J. Livny, J. A. D. Good, S. Sarkar, D. X. Zhu, R. L. Kinsella, L. A. Weiss, S. D. Solomon, M. E. Schene, M. R. Hansen, A. G. Cairns, M. Kulén, T. Wixe, A. E. G. Lindgren, E. Chorell, C. Bengtsson. K. S. Krishnan, S. J. Hultgren, C. Larsson, F. Almqvist, C. L. Stallings. PNAS, 2019, 116, 10510-10517. 5. (a) P. Engström, K. S. Krishnan, B. D. Nguyen, E. Chorell, J. Normark, J. Silver, R. J. Bastidas, M. D. Welch, S. J. Hultgren, H. Wolf-Watz, R. H. Valdivia, F. Almqvist, S. Bergström, m.Bio, 2015, 6 (1), 2304–2314. (b) J. A. D. Good, J. Silver, C. Núñez-Otero, W. Bahnan, K. S. Krishnan, O. Salin, P. Engström, R. Svensson, P. Artursson, Å. Gylfe, S. Bergström, F. Almqvist, J. Med. Chem. 2016, 59, 2094–2108. (c) J. A. D. Good, M. Kulén, J. Silver, K. S. Krishnan, W. Bahnan, C. Núñez-Otero, I. Nilsson, E. Wede, E. de Groot, Å Gylfe, S. Bergström, F. Almqvist, J. Med. Chem. 2017, 60, 9393–9399. (d) Kulén, M.; Núñez-Otero, C.; Cairns, A.G.; Silver, J.; Lindgren, A.E.; Wede, E.; Singh, P.; Vielfort, K.; Bahnan, W.; Good, J.A.; Svensson, R. Med. Chem. Comm., 2019, 10, 1966-1987. 6. P. Singh, E. Chorell, K. S. Krishnan, T. Kindahl, J. Åden, P. Wittung-Stafshede, F. Almqvist, Org. Lett. 2015, 17, 6194–6197. 7. P. Singh, D. E. Adolfsson, J. Ådén, A. G. Cairns, C. Bartens, K. Brännström, A. Olofsson, F. Almqvist, J. Org. Chem. 2019, 84, 3887–3903. 8. (a) Twilton, J.; Zhang, P.; Shaw, M.H.; Evans, R.W.; MacMillan, D.W. Nat. Rev. Chem., 2017, 1, 0052. (b) Shaw, M.H.; Twilton, J.; MacMillan, D.W. J. Org. Chem., 2016, 81, 6898-6926. (c) Bogdos, M.K.; Pinard, E.; Murphy, J.A., Beilstein J. Org. Chem., 2018. 14, 2035-2064. 9. (a) Budnikova, Y.H.; Sinyashin, O.G.D. Russ. Chem. Rev., 2015, 84, 917-951. (b) Helmchen, G.; Pfaltz, A., Acc. Chem. Res., 2000, 33, 336-345. (c) Netherton, M.R.; Fu, G.C. Org. Lett., 2001, 3, 4295-4298. (d) Surry, D.S.; Buchwald, S.L., Angew. Chem. Inte. Ed., 2008, 47, 6338-6361. 10. Chen, X.; Kopecky, D.J.; Mihalic, J.; Jeffries, S.; Min, X.,;Heath, J.; Deignan, J.; Lai, S.; Fu, Z.; Guimaraes, C.; Shen, S., J. Med. Chem., 2012, 55, 3837-3851. 26

(b) Krise, J.P.; Stella, V.J., Adv. Drug Del. Rev., 1996, 19, 287-310. (c) Moonen, K.; Laureyn, I.; Stevens, C.V. Chem. Rev., 2004, 104, 6177-6216. 11. Allcock, H.R.; Hofmann, M.A.; Ambler, C.M.; Morford, R.V.; Macromolecules, 2002, 35, 3484-3489. (b) Onouchi, H.; Miyagawa, T.; Furuko, A.; Maeda, K.; Yashima, E. J.A.C.S, 2005, 127, 2960-2965. (c) Demmer, C.S.; Krogsgaard- Larsen, N.; Bunch, L. Chem. Rev, 2011, 111, 7981-8006. 12. (a) Han, L.B.; Tanaka, M. J.A.C.S, 1996, 118, 1571-1572. (b) Kalek, M., Stawinski, J. Adv.Synth. Catal. 2011, 353, 1741-1755. Gui, Q.; Hu, L.; Chen, X.; Liu, J.; Tan, Z.; Chem.Commun. 2015, 51, 13922-13924. Zhuang, R.; Xu, J.; Cai, Z.; Tang, G.; Fang, M.; Zhao, Y., Org.Lett, 2011, 13, 2110-2113. Feng, C.G.; Ye, M.; Xiao, K.J.; Li, S.; Yu, J.Q. J.A.C.S, 2013, 135, pp.9322-9325. Yang, J.; Chen, T.; Han, L.B. J.A.C.S., 2015, 137, 1782-1785. Li, D.P.; Pan, X.Q.; An, L.T.; Zou, J.P.; Zhang, W. J. Org. Chem., 2014, 79,1850-1855. Wu, L.; Zhang, X.; Chen, Q.Q.; Zhou, A.K. 2012. Org. Biomol.Chem., 10, 7859- 7862. 13. (a) Berger, O.; Montchamp, J. L. Chem. Eur. J. 2014, 20, 12385-12388. (b) Xiang, C.B.; Bian, Y.J.; Mao, X.R.; Huang, Z. Z. J. Org.Chem. 2012. 77, 7706- 7710. (c) Huang, X.F.; Wu, Q.L.; He, J.S.; Huang, Z.Z. Org. Biomol. Chem. 2015, 13, 4466-4472. (d) Kagayama, T.; Nakano, A.; Sakaguchi, S.; Ishii, Y. Org. Lett, 2006, 8, 407-409. (e) Wang, H.; Li, X.; Wu, F.; Wan, B. Synthesis, 2012, 44, 941-945. (f) Kottmann, H.; Skarzewski, J.; Effenberger, F. Synthesis, 1987, 09, 797-801. (g) H. Kottmann, J. Skarzeweski, F. Effenberger, Synthesis 1987, 9, 797 – 801. 14. (a) Luo, K.; Yang, W.C.; Wu, L.; Asian J. Org. Chem. 2017, 6, 350-367. (b) Cai, B.G.; Xuan, J.; Xiao, W.J. Sci. Bull. 2019, 64, 337-350. 15. Shaikh, R.S.; Düsel, S.J.; König, B.. ACS Catal. 2016, 6, 8410-8414. 16. Zhao, Z.; Min, Z.; Dong, W.; Peng, Z.; An, D. Synth. Commun.2016, 46, 128- 133. 17. Shaikh, R.S.; Ghosh, I.; König, B. Chem. Eur. J. 2017, 23, 12120-12124. 18. Dreher S. D. React.Chem.Eng., 2019, 4, 1530 19. Boström, J.; Brown, D.G.; Young, R.J.; Keserü, G.M. Nat. Rev. Drug Discov. 2018, 17, 709-727. 20. Zuo, Z.; Ahneman, D.T.; Chu, L.; Terrett, J.A.; Doyle, A.G.; MacMillan, D.W. 2014, Science, 345, 437-440. 21. Oderinde, M.S.; Johannes, J.W. Org. Synt. 2017, 77-92. 22. Marma, M. S.; Khawli, L. A.; Harutunian, V.; Kashemirov, B. A.; McKenna, C. E., J. Fluor. Chem., 2005, 126, 1467-1475. 23 Evans, M.L.; Chorell, E.; Taylor, J.D.; Åden, J.; Götheson, A.; Li, F.; Koch, M.; Sefer, L.; Matthews, S.J.; Wittung-Stafshede, P. Almqvist, F. Chapman, M.R. Mol. cell, 2015, 57,445-455.

Appendix 1

1 Compound 17a, H-NMR (400 MHz) (CDCl3):

CF3

S N

CO2Me

1 Compound 17b, H-NMR (400 MHz) (CDCl3):

S N

CO2Me

1 Compound 17c, H-NMR (400 MHz) (CDCl3):

OMe S N

CO2Me

1 Compound 20a, H-NMR (400 MHz) (CDCl3):

O O

1 Compound 20b, H-NMR (400 MHz) (CDCl3):

CF3

O O

1 Compound 20c, H-NMR (600 MHz) (CDCl3):

Cl HO

O O

1 Compound 21a, H-NMR (400 MHz) (CDCl3):

CF3

S N

O CO2Me

1 Compound 21b, H-NMR (600 MHz) (CDCl3):

S N

O CO2Me

1 Compound 21c, H-NMR (400 MHz) (CDCl3):

O S N

O CO2Me

1 Compound 21d, H-NMR (400 MHz) (CDCl3):

F3C S N

O CO2Me

1 Compound 21e, H-NMR (600 MHz) (CDCl3):

Cl S N

O CO2Me

Compound 29a,1H-NMR (400 MHz) (CD3)2SO:

S N

O N O H

1 Compound 29b, H-NMR (400 MHz) (CDCl3):

S N

O N O H

1 13 Compound 27a, H-NMR (400 MHz) and C-NMR (100 MHz) (CDCl3):

F3C

S N (EtO)2OP O CO2Me

1 13 Compound 27b, H-NMR (400 MHz) and C-NMR (151 MHz) (CDCl3):

S N (EtO)2OP O CO2Me

1 13 Compound 27c, H-NMR (600 MHz) and C-NMR (151 MHz) (CDCl3):

O S N (EtO)2OP O CO2Me

1 13 Compound 27d, H-NMR (600 MHz) and C-NMR (100 MHz) (CDCl3):

S F3C N (EtO)2OP O CO2Me

1 13 Compound 30a, H-NMR (400 MHz) and C-NMR (151 MHz) (CDCl3):

S N (EtO)2OP O N O H

1 13 Compound 30b, H-NMR (600 MHz) and C-NMR (151 MHz) (CDCl3):

S N (EtO)2OP O N O H

1 13 Compound 30c, H-NMR (600 MHz) and C-NMR (151 MHz) (CDCl3):

O S N (EtO)2OP F O N O H

1 13 Compound 28a, H-NMR (600 MHz) and C-NMR (151 MHz) (CD3)2SO):

F3C

S N (HO)2OP O CO2H

1 13 Compound 28b, H-NMR (600 MHz) and C-NMR (151 MHz) (CD3)2SO):

S N (HO)2OP O CO2H

1 13 Compound 28c, H-NMR (600 MHz) and C-NMR (151 MHz) (CD3)2SO):

O S N (HO)2OP O CO2H

1 13 Compound 28d, H-NMR (600 MHz) and C-NMR (151 MHz) (CD3)2SO):

S F3C N (HO)2OP O CO2H

Appendix 2

Procedure for the synthesis of photocatalyst (Ir[dF(CF3)ppy]2(dtbpy))PF6 ( 26):

Aryl chloride 23 (1.0 eq.), Boronic acid 22 (1.12eq.) and NaHCO3 aq. (2M) (2.25eq.) were taken in a dry round bottom flask followed by the addition of toluene/benzene (17 mL/23 mL) and the mixture was degassed by bubbling N2 for 15 min. Then, Pd(PPh3)4 was added and the mixture was degassed again with N2 for 15 min. The reaction was then refluxed for 72 h. TLC (DCM/Heptane 1:1) showed complete conversion. The reaction mixture was then transferred to a separatory funnel, diluted with DCM and washed with water and brine. The aq. layer was extracted 3 times with DCM and the combined organic layers were dried over Na2SO4 and concentrated under vacuum. The crude was loaded to silica and purified with DCM/heptane starting with 20-70% step gradient. The broad peak eluted at around 40-50% DCM was collected to afford white solid. 3.1g 22 was converted into 2.88 g of 24 (65% yield).

In the next step, in an oven dried microwave vial equipped with a magnetic stir bar IrCl3·xH2O (1.0 eq.), cyclometalating ligand (phenyl pyridyl) 24 (8.0 eq.), and ethylene glycol (10 mL). The vial was sealed and pre stirred for 1 min prior to heating under microwave irradiation (200 °C, 50 min) at atmospheric pressure. Thereafter the mixture was allowed to cool to room temperature, the dative ligand (bipyridyl 25 (1.5 eq.) was added and the vial was heated under microwave irradiation (200 °C, 30 min) at atmospheric pressure. After being cooled to room temperature, the reaction mixture was diluted with deionized H2O (25 mL) and extracted with EtOAc and once with hexane (50 mL). The aqueous layer was extracted with ethyl acetate, and the combined ethyl acetate extract layers were collected, filtered, dried over Na2SO4, and concentrated in vacuo. Deionized H2O (60 mL) was added to the mixture to generate a yellow solution with free-flowing red-orange solids, to which aqueous ammonium hexafluorophosphate (4.0 g in 40 mL of deionized H2O) was added to give a yellow solid. The resulting precipitate was collected and washed with cold deionized H2O (30 mL) and cold diethyl ether (30 mL). Finally, the precipitate was taken up in acetone and dried in vacuo. The desired product was afforded after recrystallization with acetone and diethyl ether (1:4) at low temperatures. 1.28 g of 24 was converted into 500 mg of 26 1 (75% yield). H NMR (400 MHz, (CD3)2CO) δ 8.95 (d, J = 1.8 Hz, 2H), 8.62 (d, J = 8.9 Hz, 2H), 8.41 (d, J = 8.8 Hz, 2H), 8.19 (d, J = 5.9 Hz, 2H), 7.83-7.81 (m, 4H), 6.90 – 6.84 (m, 2H), 5.98 (dd, J = 8.4, 2.3 Hz, 2H), 1.44 (s, 18H).

1 Compound 26, H-NMR (400 MHz) and ((CD3)2CO):