Development of New Novel Bacterial Topoisomerase Inhibitors As Promising Antibiotics with a 5-Amino-1,3-Dioxane Linker Moiety DI

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Development of New Novel Bacterial Topoisomerase Inhibitors as Promising Antibiotics with a 5-Amino-1,3-dioxane Linker Moiety

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Linsen Li

Graduate Program in Pharmaceutical Sciences

The Ohio State University

2019

Dissertation Committee:

Mark J Mitton-Fry, Ph.D., Advisor

Karl A Werbovetz, Ph.D.

Mireia Guerau-de-Arellano, Ph.D.

James R Fuchs, Ph.D.

Abstract

Multidrug-resistant bacteria (MDR) have become one of the greatest threats to human beings. Regardless of the mechanism of action involved, resistance has eventually developed after the clinical use of each class of antibiotics. Thus, new antibiotic classes with unprecedented mechanisms of action are in urgent demand. With a distinct mechanism of action, Novel Bacterial Topoisomerase Inhibitors (NBTIs) represent a promising new class of antibiotics. The recent development of NBTIs has been encouraging. Several

NBTIs have processed to clinical trials, and one NBTI (Gepotidacin) has successfully completed Phase 2 clinical trials. However, the cardiac toxicity from the inhibition of hERG K+ channels is one of the major challenges in the discovery of NBTIs.

In this work, a 5-amino-1,3-dioxane linker moiety was incorporated in new NBTIs to decrease hERG inhibition, which was demonstrated by comparison with several structure- matched pairs with other linker moieties. A variety of NBTIs with a 5-amino-1,3-dioxane linker have been synthesized and evaluated. Many of these newly synthesized NBTIs showed potent antibacterial activity, covering methicillin-resistant Staphylococcus aureus

(MRSA) and fluoroquinolone-resistant strains. Some of these new NBTIs displayed dual inhibition against both DNA gyrase and topoisomerase IV of S. aureus, but they targeted

DNA gyrase more potently than topoisomerase IV. These new NBTIs presented low toxicity to human cells and minimal inhibition of human topoisomerase IIα. Additionally, several new NBTIs revealed favorable hERG profiles. With promising preclinical

ii attributes, some of these NBTIs have the potential to be evaluated in in vivo experiments and can serve as the starting point for further development of new NBTIs.

iii

Dedication

This document is dedicated to my grandparents.

Acknowledgements

It’s like a formality to express thanks to the advisor in the dissertation of each doctoral student. However, I would like to give my sincere appreciation and admiration to my advisor, Dr. Mark Mitton-Fry. I joined the Mitton-Fry research group and became the first student in the lab in the summer of 2016. Then I found this mid-aged man very interesting.

Dr. Mitton-Fry is always walking with his fast split steps, enjoying his time in front of his research hood. It’s very easy to tell his brilliant intelligence at most times, but he was even not able to set up an old iPhone, although he tried hard to learn from his young daughter.

During the past years, his expertise, cautious and discreet attitude toward research, and passion for work have always inspired me tremendously. I feel very lucky to have Dr.

Mitton-Fry as my advisor in my Ph.D. program.

I’m grateful to the medicinal chemistry program in the College of Pharmacy for offering me the opportunity to study and work here with many great scholars from the whole world.

I’m grateful to Dr. Chenglong Li as my research advisor for my first three years in the program. I learned many useful computational tools for drug discovery and got training in synthetic chemistry in Dr. Chenglong Li’s lab. I’m also grateful to Dr. James Fuchs, Dr.

Karl Werbovetz and Dr. Mireia Guerau-de-Arellano for serving as my committee members.

They have been always willing to help and have given me lots of beneficial suggestions. I would like to thank Dr. Craig McElroy for assistance with the research instruments. I’m also thankful to all my lab-mates in Dr. Mitton-Fry’s lab and Dr. Chenglong Li’s lab. These interesting people have created a joyful environment in the lab and helped a lot.

I would like to thank all my friends met in the Ohio State University. There are so many fantastic things around campus: the white bass and rainbow trout swimming under harmonious spring breeze, the soccer balls dribbling upon lush green lawn, the snowboard flipping from peak of white mountains, and so on. It was my friends that painted these fantastic things into my memory, which made my Ph.D. program a wonderful journey. I would like to thank my roommates in Nanjing University and intimate friends, Dr. Lichen

Liu, Hao Li, Dr. Mufan Li and Jinping Liu. Regardless of the long distance apart each of us, our daily conversations gave me not only broad insight into chemistry, but also more comprehensive thoughts on the world.

Finally, I would like to thank my family. It was their love and selfless support that made the completion of this thesis possible.

Vita

2008 – 2012………………..B.S. Chemistry, Nanjing University

2012 – 2013………………..Research Assistant, Department of Chemistry, Tsinghua

University

2013 – present …………….Graduate Teaching Associate, Graduate Research

Associate, Division of Medicinal Chemistry &

Pharmacognosy, The Ohio State University

Publications

Li L, Okumu AA, Nolan S, English A, Vibhute S, Lu Y, Hervert-Thomas K, Seffernick JT, Azap

L, Cole SL, Shinabarger D, Koeth LM, Lindert S, Yalowich JC, Wozniak DJ, Mitton-Fry MJ.

1,3-Dioxane-Linked Bacterial Topoisomerase Inhibitors with Enhanced Antibacterial Activity and Reduced hERG Inhibition. ACS Infect Dis 2019, 5, 1115-1128.

Li L, Okumu AA, Nolan S, Li Z, Karmahapatra S, English A, Yalowich JC, Wozniak DJ, Mitton-

Fry MJ. Synthesis and Anti-staphylococcal Activity of Novel Bacterial Topoisomerase Inhibitors with a 5-Amino-1,3-dioxane Linker Moiety. Bioorg Med Chem Lett 2018, 28, 2477-2480.

Webb LM, Amici SA, Jablonski KA, Savardekar H, Panfil AR, Li L, Zhou W, Peine K,

Karkhanis V, Bachelder EM, Ainslie KM, Green PL, Li C, Baiocchi RA, Guerau-de-Arellano M.

PRMT5-Selective Inhibitors Suppress Inflammatory T Cell Responses and Experimental

Autoimmune Encephalomyelitis. J Immunol 2017, 198, 1439-1451. vii

Li L, Niu Z, Cai S, Zhi Y, Li H, Rong H, Liu L, Liu L, He W, Li Y. A PdAg bimetallic nanocatalyst for selective reductive amination of nitroarenes. Chem Commun 2013, 49,

6843-6845.

Zhang Q, Cai S, Li L, Chen Y, Rong H, Niu Z, Liu J, He W, Li Y. Direct Syntheses of

Styryl Ethers from Benzyl Alcohols via Ag Nanoparticle-Catalyzed Tandem Aerobic

Oxidation. ACS Catalysis 2013, 3, 1681-1684.

Li H, Li L, Li Y. The Electronic Structure and Geometric Structure of Nanoclusters as

Catalytic Active Sites. Nanotechnology Reviews 2013, 2, 515-528.

Fields of Study

Major Field: Pharmacy (Pharmaceutical Sciences)

viii

Table of Contents

Abstract ...... ii

Dedication ...... iv

Acknowledgements ...... v

Vita ...... vii

List of Tables ...... xiii

List of Figures ...... xiv

List of Scheme ...... xvi

Chapter 1. Brief Overview of Antibiotics and Novel Bacterial Topoisomerase Inhibitors ...... 1

1.1 Introduction ...... 1

1.2 Typical Classes of Antibiotics ...... 1

1.2.1 Cell wall antibiotics ...... 2

1.2.1.1 β-lactam antibiotics ...... 3

1.2.1.1 Glycopeptide antibiotics ...... 5

1.2.2 Bacterial ribosome antibiotics ...... 7

1.2.2.1 Aminoglycoside antibiotics ...... 8

1.2.2.2 Macrolide antibiotics ...... 10

1.2.2.3 Tetracycline antibiotics ...... 11

1.2.2.4 Oxazolidinone antibiotics ...... 11

1.2.3 Cell membrane antibiotics ...... 12

1.2.4 Folate biosynthesis antibiotics ...... 14

1.2.5 DNA transcription antibiotics ...... 15

1.2.6 DNA replication antibiotics ...... 16

1.3 Quinolones as Antibiotics ...... 17

1.3.1. Mechanism of action for quinolones ...... 18

1.3.2. Resistance to quinolones ...... 21

1.3.3. Adverse effect of quinolones ...... 22

1.4 Novel Bacterial Topoisomerase Inhibitors ...... 23

1.4.1. Typical structures of NBTIs ...... 24

1.4.2. Mechanism of action for NBTIs ...... 24

1.4.3. Previous chemical efforts to develop NBTIs ...... 26

1.4.3.1 NBTIs with aminopiperidine linker ...... 27

1.4.3.2 NBTIs with tetrahydroindazole linker ...... 31

1.4.3.3 NBTIs with carboxypiperidine Linker ...... 32

1.4.3.4 NBTIs with aminocyclohexane linker ...... 34

1.4.3.5. NBTIs with tetrahydropyran linkers ...... 35

1.4.3.6. NBTIs with oxabicyclooctane linkers ...... 37

1.4.3.7. Gepotidacin...... 40

1.5 Conclusions ...... 41

1.6 References ...... 42

Chapter 2. Development of 5-amino-1,3-dioxane Linked NBTIs with 6-methoxyquinoline DNA- binding Moiety ...... 53

2.1 Abstract ...... 53

2.2 Introduction ...... 53

2.3 Results and Discussion ...... 58

2.3.1 Synthesis ...... 58

2.3.1.1 Synthesis of NBTIs possessing a 6-methoxyquinoline DNA-binding moiety ...... 58

2.3.1.2 Synthesis of 3,4-dihydro-2H-pyrano[2,3-c]pyridine-6-carbaldehyde ...... 61

2.3.1.3 Synthesis of 3-oxo-3,4-dihydro-2H-pyrido[3,2-b][1,4]thiazine-6- carbaldehyde ..... 62

2.3.1.4 Synthesis of 3-oxo-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazine-6- carbaldehyde ..... 63

2.3.1.5 Synthesis of vinyl 6-methoxyquinoline moiety series ...... 64

2.3.1.6 Synthesis of 6-methoxyquinoline analogs with a cyclohexane linker ...... 65

2.3.1.7 Synthesis of 6-methoxyquinoline analogs with a piperidine linker ...... 66

2.3.2 Biological evaluation of NBTIs possessing a 5-amino-1,3-dioxane linker ...... 67

2.3.2.1 Minimum inhibitory concentrations ...... 67

2.3.2.2 Targeted enzyme inhibition ...... 73

2.3.2.3 Preliminary in vitro safety evaluation ...... 74

2.4 Conclusions ...... 77

2.5 Experimental Section...... 78

2.5.1 Chemistry part ...... 78

2.5.2 Biological evaluation assays ...... 116

2.6 Reference ...... 118

Chapter 3. Optimization of 5-amino-1,3-dioxane Linked NBTIs ...... 121

3.1 Abstract ...... 121

3.2 Introduction ...... 121

3.3 Results and Discussion ...... 124

3.3.1 Synthesis ...... 124

3.3.1.1 Synthesis of N-linked quinoxalinone analogs ...... 125

3.3.1.2 Synthesis of 7-fluoro-2-methoxy-1,5-naphthyridine analogs ...... 127

3.3.1.3 Synthesis of 1-(3-fluoro-6-methoxy-1,5-naphthyridin-4-yl)ethan-1-ol analogs ..... 128

3.3.1.4 Synthesis of 2-(5-(((6,7-dihydro-[1,4]oxathiino[2,3-c]pyridazin-3-yl) methyl)amino)- trans-1,3-dioxan-2-yl)-1-(3-fluoro-6-methoxyquinolin-4-yl)ethan-1-ol ...... 129

3.3.1.5 Synthesis of 2-(7-fluoro-2-methoxyquinolin-8-yl)ethan-1-ol analogs ...... 131

3.3.1.6 Efforts to optimize the synthesis of NBTIs with C-2 hydroxyl linker...... 133

3.3.2 Biological evaluation of new NBTI analogs ...... 135

3.3.2.1 Minimum inhibitory concentrations ...... 135

3.3.2.2 Targeted enzyme inhibition ...... 140

3.3.2.3 Spontaneous Frequency of Resistance ...... 143

3.3.2.4 Preliminary in vitro safety evaluation ...... 144

3.4 Conclusions ...... 146

3.5 Experimental Section...... 147

3.5.1 Chemistry part ...... 147

3.5.2 Biological evaluation assays ...... 179

3.6 Reference ...... 182

Bibliography ...... 185

Appendix. Copies of 1H NMR, 13C NMR and MS of Desired Compounds ...... 202

xii

List of Tables

Table 2.1. Minimal inhibitory concentrations (MICs) for 1,3-dioxane linked 6-methoxyquinolines

...... 69

Table 2.2 MICs for 6-methoxyquinoline series with vinyl, cyclohexane, piperidine linker ...... 71

Table 2.3 MICs for selected compounds in MSSA and MRSA cellular inhibitory experiments .. 72

Table 2.4 Target inhibition of selected compounds ...... 74

Table 2.5 In vitro safety evaluation data of selected NBTIs ...... 75

Table 3.1．MICs for selected compounds in MSSA and MRSA cellular inhibitory experiments

...... 136

Table 3.2 MICs for ciprofloxacin-resistant strain SA-3527 and 1490-08 ...... 138

Table 3.3 Broad spectrum antibacterial activity of select NBTIs ...... 139

Table 3.4 Target inhibition of new analogs...... 141

Table 3.5 Inhibition of wild-type and fluoroquinolone-resistant (S84L) DNA gyrase ...... 142

Table 3.6 Spontaneous Frequency of Resistance for representative compounds ...... 143

Table 3.7 In vitro safety assessment of potent NBTIs ...... 145

xiii

List of Figures

Figure 1.1 Structures of representative β-lactam antibiotics ...... 3

Figure 1.2 β-lactamase inhibitors ...... 4

Figure 1.3 Structure of vancomycin ...... 6

Figure 1.4 Structures of representative aminoglycosides: 2-deoxystreptamine, streptomycin, kanamycin A, spectinomycin, neomycin C, gentamicin ...... 9

Figure 1.5 Structures of representative macrolides antibiotics: erythromycin and telithromycin . 10

Figure 1.6 Structures of representative tetracycline antibiotics...... 11

Figure 1.7 Structures of linezolid and tedizolid ...... 12

Figure 1.8 Structures of colistin and daptomycin ...... 13

Figure 1.9 Structures of sulfadoxine, sulfamethoxazole, sulfafurazole and trimethoprim ...... 14

Figure 1.10 Structures of rifampicin, rifaximin and fidaxomicin ...... 15

Figure 1.11 ATPase inhibitor: novobiocin ...... 16

Figure 1.12 Representative quinolones of first, second, third, and fourth generations ...... 18

Figure 1.13 Mechanism relaxation of supercoiling by DNA gyrase [101] ...... 20

Figure 1.14 Structures of representative NBTIs ...... 24

Figure 1.15 Typical structure of NBTIs represented by GSK299423 and NXL101 ...... 25

Figure 1.16 2.1 Å gyrase GyrB27-A56 complex with GSK299423...... 26

Figure 1.17 NBTIs with aminopiperidine linker and bicyclic DNA-binding domain ...... 29

Figure 1.18 NBTIs with aminopiperidine linker and tricyclic DNA-binding domains ...... 30

Figure 1.19 NBTIs with aminopiperidine linker and fused tricyclic DNA-binding domain ...... 30

Figure 1.20 NBTIs with tetrahydroindazole linker ...... 32

xiv

Figure 1.21 NBTIs with carboxypiperidine linker ...... 33

Figure 1.22 NBTIs with aminocyclohexane linker ...... 34

Figure 1.23 NBTIs with tetrahydropyran linkers ...... 36

Figure 1.24 NBTIs with oxabicyclooctane linkers ...... 39

Figure 1.25 Structure of gepotidacin ...... 41

Figure 2.1 Structures of AM-8085, ACT-387042, NBTI-5463, gepotidacin and GSK966587 .... 55

Figure 2.2 Typical NBTI structure: GSK299423 ...... 56

Figure 2.3 Selective PAK1 inhibitor G-5555 and our representative NBTI, Compound 9 ...... 57

Figure 3.1 AstraZeneca NBTIs with N-linked quinoxalinone moiety ...... 122

Figure 3.2 Merck NBTIs: AM8085 and AM8191. Actelion NBTIs: Actelion-5 and ACT-387042.

...... 123

Figure 3.3 Structures of new 5-amino-1,3-dioxane linked NBTIs ...... 124

List of Scheme

Scheme 2.1 Synthesis of NBTIs possessing a 6-methoxyquinoline DNA-binding moiety...... 58

Scheme 2.2 Structure of the library of compounds possessing a 6-methoxyquinoline moiety and a

5-amino-1,3-dioxane linker...... 60

Scheme 2.3 Synthesis of 3,4-dihydro-2H-pyrano[2,3-c]pyridine-6-carbaldehyde...... 61

Scheme 2.4 Synthesis of 3-oxo-3,4-dihydro-2H-pyrido[3,2-b][1,4]thiazine-6- carbaldehyde.. ... 62

Scheme 2.5 Synthesis of 3-oxo-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazine-6- carbaldehyde...... 63

Scheme 2.6 Synthesis of vinyl compounds (86 and 87) possessing a 6-methoxyquinoline moiety..

...... 64

Scheme 2.7 Synthesis of 6-methoxyquinoline analogs with a cyclohexane linker...... 65

Scheme 3.1 Synthesis of N-linked quinoxalinone analogs...... 125

Scheme 3.2 Synthesis of 7-fluoro-2-methoxy-1,5-naphthyridine analogs...... 127

Scheme 3.3 Synthesis of 1-(3-fluoro-6-methoxy-1,5-naphthyridin-4-yl)ethan-1-ol analogs...... 128

Scheme 3.4 Synthesis of 2-(5-(((6,7-dihydro-[1,4]oxathiino[2,3-c]pyridazin-3-yl) methyl)amino)- trans-1,3-dioxan-2-yl)-1-(3-fluoro-6-methoxyquinolin-4-yl)ethan-1-ol...... 129

Scheme 3.5 Synthesis of 2-(7-fluoro-2-methoxyquinolin-8-yl)ethan-1-ol analogs...... 131

Scheme 3.6 Proposed alternative synthetic pathway to 2-(5-amino-trans-1,3-dioxan-2-yl)-1-(7- fluoro-2-methoxyquinolin-8-yl)ethan-1-ol primary amine ...... 133

xvi

Chapter 1. Brief Overview of Antibiotics and

Novel Bacterial Topoisomerase Inhibitors

1.1 Introduction

The global spread of multidrug-resistant bacteria (MDR) is one of the greatest threats to human beings, not only for public health but also for political and economic development.[1] Currently around 0.7 million people lose their lives per annum due to antibiotic-resistant infections, and it has been estimated that resistant infections will cause 10 million deaths per year by 2050.[2] Methicillin-resistant Staphylococcus aureus (MRSA) has become very common around the world and is a leading cause of bacterial infections [3]. Sadly, the situation has become even worse with the continual dissemination and discovery of new multidrug resistant “superbugs”. For example, a MDR strain of Escherichia coli resistant to polymyxin E was discovered recently [4]. Many factors contribute to the crisis of antimicrobial resistance, including the overuse of antibiotics in health care, agriculture and the environment [5]. Bacteria can develop resistance to any antimicrobial reagents through a variety of different mechanisms [6]. Accordingly, two general measures should be taken in order to combat this crisis: one is to reduce the usage of antimicrobials so that the current antibiotic stock can last longer, and the other is to increase the supply of new antibiotics with efficacy against resistant bacteria. [2] From the perspective of the latter, it is necessary to understand the mechanisms of action and resistance of current antimicrobial reagents in order to develop new therapeutics.

1.2 Typical Classes of Antibiotics

Since the discovery of penicillin G (Fig 1.1) in 1928 [7], various antibiotics have been

1 developed with the following primary mechanisms: interference with cell wall synthesis, dysfunction of bacterial protein synthesis, disturbance of folate biosynthesis, disruption of lipid membrane integrity, and inhibition of DNA replication or transcription. [8]

1.2.1 Cell wall antibiotics

The cell wall of bacteria provides rigidity to bacterial cells and is essential to bacterial survival. Peptidoglycan serves as the structural unit for the bacterial cell wall. It consists of repeating N-acetylglucosamine (NAG) linked with N-acetylmuramic acid (NAM, β- 1 to 4 glycosidic linkages). Attached to the NAM component is a peptide chain of three to five amino acids that hangs from each peptidoglycan strand. For Gram-positive bacteria, the peptide chain is a pentapeptide of L-Ala–D-Glu–L-Lys–D-Ala–D-Ala. For Gram- negative bacteria, the third amino acid (L-lysine) is replaced with diaminopimelic acid (DAP). Generally, peptidoglycan strands are cross-linked by the peptide chain, forming the three-dimensional architecture of the bacterial cell wall. The cross-linking occurs between the carboxylic acid group of D-Ala at position 4 and the amino group of the diamino acid at position 3 of two adjacent peptide chains through a short peptide bridge. [9] This process is catalyzed by transpeptidase. Besides the difference in amino acid components in the cross-linking peptide chain of the cell wall, Gram-negative bacteria contain an additional thick outer membrane which is an asymmetric bilayer of lipopolysaccharides and phospholipids. The low permeability of the outer membrane to diverse compounds provides Gram-negative bacteria with resistance to many antibiotics which are effective against Gram-positive bacteria [10]. The resistance is further reinforced by the powerful efflux capacity of multidrug efflux transporters in Gram-negative bacteria, which keeps cytoplasmic antibiotics from achieving efficacious concentrations. Consequently, the discovery of effective Gram-negative antibiotics is particularly challenging and has drawn substantial efforts [10].

As recently reviewed, diverse enzymes and other components involved in bacterial cell wall construction have been targeted by different classes of antibiotics [11]. For example, D-Ala–D-Ala ligase and D-Ala racemase are inhibited by D-cycloserine, and

2 transglycosylase is the target of the moenomycin family. Full details are not listed here; instead, a highlight is given to those drugs that have played a particularly significant role in history and their associated mechanisms of action.

1.2.1.1 β-lactam antibiotics

Figure 1.1 Structures of representative β-lactam antibiotics

As representative antibiotics targeting the bacterial cell wall, the β-lactam antibiotics (Fig 1.1), particularly the penicillin and cephalosporin subfamilies, occupy more than half of the market share of all commercial antibiotics [12]. The targets of β-lactam antibiotics are so-called penicillin binding proteins (PBPs), which are mostly peptidoglycan transpeptidases in cell wall biosynthesis, especially serine-type D-alanyl-D-alanine transpeptidase. The 4-membered cyclic amide (β-lactam ring) is the common structure of all β-lactam antibiotics, and it mimics the acyl-D-Ala-D-Ala portion of the peptide terminus attached to each peptidoglycan strand. The active serine residue in the catalytic site of the penicillin binding proteins attacks the carbonyl group of the β-lactam ring, cleaving the ring and forming a covalent and irreversible acyl-enzyme complex. [11, 13] As a result, the β-lactam compounds completely block the active site of the transpeptidase, which inhibits the crosslinking of peptidoglycan strands and compromises the integrity of

3 bacterial cell wall.

Figure 1.2 β-lactamase inhibitors

The most common form of resistance to the β-lactam class comes from the production of β-lactamase enzymes. β-lactamase enzymes catalyze the hydrolysis of the β-lactam ring of the β-lactam antibiotics. These enzymes can hydrolyze all different types of β-lactam antibiotics before they bind to the penicillin binding proteins. There are two general types of β-lactamases: serine β-lactamases and metallo-β-lactamases. The Ambler molecular classification divides them into four classes: three for serine β-lactamases (classes A, C, D) and one for metallo-β-lactamases (class B). [14] The serine β-lactamases catalyze the hydrolysis of the β-lactams through the attack of an active site serine residue on the β- lactam and subsequent hydrolysis of the resulting acyl enzyme intermediate. In contrast, the metallo-β-lactamases effect the hydrolysis of β-lactams through the attack of a hydroxide ion that is stabilized by a Zn2+ ion coordinated to histidine/cysteine/aspartic acid residues in the active site [14]. The introduction of β-lactamase inhibitors is consequently a useful strategy to combat resistance from β-lactamase.

The β-lactamase inhibitors bind to the active site of serine β-lactamase with high affinity, but they do not undergo rapid hydrolysis like the β-lactams. Combined with β- lactam antibiotics, β-lactamase inhibitors have been developed as an efficacious approach to antimicrobial therapy [14]. Clavulanic acid (Fig 1.2) was the first β-lactamase inhibitor introduced into clinical use. In the 1970s, it was launched together with amoxicillin (named 4 as Augmentin) or ticarcillin (named as Timentin) [15]. In the 1980s, sulbactam (Fig 1.2) was combined to form ampicillin/sulbactam (named as Unasyn) [16]. Very recently, the combination of tazobactam (Fig 1.2) and ceftolozane (named as Zerbaxa) [17] was approved to treat complicated urinary tract infections and complicated intra-abdominal infections. Additionally, three β-lactamase inhibitors lacking a β-lactam structure were approved by FDA very recently. Avibactam (Fig 1.2), together with ceftazidime (named as Avycaz), was approved to treat complicated urinary tract and complicated intra- abdominal infections caused by antibiotic-resistant pathogens in 2015 [18]. Vaborbactam, a serine β-lactamase inhibitor which incorporates a boronic acid moiety, (Fig 1.2) was approved in combination with meropenem (named as Vabomere) for complicated urinary tract infections and pyelonephritis in 2017 [19]. Very recently in 2019, FDA accepted the review of a new drug application (NDA) for Merck’s investigational combination therapy, relebactam (Fig 1.2)/imipenem and cilastatin [20]. Nevertheless, the battle against β- lactamase resistance has always been accompanied by the evolution of new β-lactamase enzymes. β-lactamases resistant to the latest inhibitors have been discovered in clinical isolates [21], which illustrates the need for new effective strategies against β-lactamases and for novel antibiotics with distinct mechanisms.

1.2.1.1 Glycopeptide antibiotics

Glycopeptides are another significant class of antibiotics which inhibit the synthesis of bacterial cell walls. They can bind to the acyl-D-alanyl-D-alanine moiety of the peptidoglycan terminus, preventing the polymerization of new peptidoglycan as the backbone strands of the cell wall [22]. If bacterial cell wall was the Great Wall, the glycopeptide antibiotics would sequester the “bricks” and prevent their incorporation into the wall. Unlike other antibiotics targeting the enzymes of bacterial cell wall biosynthesis, glycopeptides block the building substrates of the bacterial cell wall, which avoids the potential for resistance through the evolution of mutant enzymes, thereby potentially reducing the frequency of resistance. This advantage was exemplified by vancomycin (Fig 1.3), the first glycopeptide antibiotic in clinical use. Vancomycin is widely used as a “drug of last resort” against a number of multidrug-resistant infections, especially MRSA [23]. 5

In early studies, resistance to vancomycin was so difficult to induce that it was hypothesized that the development of resistance to vancomycin would be impossible [24]. Indeed, no vancomycin-resistant pathogens were documented in the first 20 years of its clinical use [25].

Figure 1.3 Structure of vancomycin

However, vancomycin-resistant pathogens were first isolated from patients in the 1980s [26]. After that, a number of unsuccessful cases of vancomycin therapy were reported [27]. High-level resistance to vancomycin was also reported in 2002 [27]. Resistance to vancomycin originated from the expression of several genes encoding proteins that change the building substrates of the bacterial cell wall. These resistant proteins can remodel the terminus of cell wall precursors from a D-Ala–D-Ala moiety to D-Ala–D-Ser or D-Ala–D-Lac moiety, thus impairing the binding affinity of vancomycin to its target [28]. The subsequent spread of vancomycin resistance has promoted the development of second-generation glycopeptide antibiotics. Three semi-synthetic glycopeptides, telavancin, oritavancin and dalbavancin, have been approved for clinical use. These semi-synthetic glycopeptides possess increased potency against vancomycin- resistant strains and favorable pharmacological properties, such as reduced dosing frequency. However, resistance has also been observed via a similar mechanism to

6 vancomycin [29]. Further efforts to develop new derivatives of glycopeptide antibiotics are in demand and on the way [30].

1.2.2 Bacterial ribosome antibiotics

Inhibitors of the bacterial ribosome represent another large class of antibiotic targets. The structure of the bacterial ribosome is sufficiently different from that of mammalian ribosomes to make the bacterial ribosome an ideal target for pharmacological agents. The ribosome provides a platform for tRNAs and mRNAs in order to translate the genetic information of mRNA into proteins [31]. Bacterial peptide synthesis is initiated from the formation of a 70S ribosome complex from a 30S subunit (ribosomal RNAs and proteins), a 50S subunit (ribosomal RNAs and proteins) and the start codon site of mRNA. [32] In the elongation stage, peptidyl tRNA delivers the amino acid according to the mRNA codon into the ribosome complex to elongate the desired peptide chain, and the ribosome then moves to the next mRNA codon site. Peptide chain synthesis is completed when a stop codon is encountered; the polypeptide chain is then released and the 70S ribosome-mRNA complex is disassembled. [32] There are three tRNA binding sites in the 70S ribosome complex: A-site, P-site and E-site. The A-site binds to the new incoming tRNA carrying the corresponding amino acid. The P-site is occupied by the tRNA carrying polypeptide chain during elongation stage or the initiating tRNA during initiation. The E-site is for the outgoing deacylated tRNA to exit the ribosome complex. During the translation process, those peptidyl-tRNAs go through the A-site, P-site and E-site consecutively, extending the desired peptide chain with the corresponding amino acids [33]. Two significant functional centers are on the ribosome complex: the peptidyltransferase center and the decoding center. The peptidyltransferase center is located on the 50S subunit and catalyzes the formation of the peptide bond. The decoding center is in the A-site and 30S subunit, and ensures that the incoming peptidyl-tRNA has the correct amino acid according to the mRNA codon. [33] Diverse classical antibiotics binding to different functional domains of the bacterial ribosome, including the 50S subunit, 30S subunit, peptidyltransferase center and decoding center, have been used in the clinic. These antibiotics include aminoglycosides, macrolides, tetracyclines, and oxazolidinones. Nevertheless, resistance 7 to these ribosome-targeting antibiotics has been widespread and extensively documented [34].

1.2.2.1 Aminoglycoside antibiotics

Aminoglycosides are one of the major types of antibiotics widely used around the world. In general, aminoglycosides have a broad antimicrobial spectrum, with especially excellent activity against Gram-negative bacteria [35]. The first aminoglycoside was streptomycin (Fig 1.4), which was discovered in 1943 and used to treat a variety of bacterial infections, including tuberculosis [35]. Additional aminoglycosides (Fig 1.4) from natural products were discovered, including neomycin, kanamycin, and gentamicin. In the 1970s, semisynthetic derivatives from older aminoglycosides were brought to the market [36], all of which contain a 2-deoxystreptamine moiety (Fig 1.4). The mechanism of action for aminoglycosides is to bind to the decoding center in the 30S subunit. More specifically, normal binding of a tRNA to the A-site in ribosome assembly has two steps. Before binding, the A-site exists in a conformationally “off” state; the first step is to recognize the corresponding tRNA and bind to it in a fast equilibrium reaction, which results in a conformational rearrangement in the A-site. The second step is a tight binding involving conformational changes of the A-site into an “on” state that enables a precise fit of the tRNA [37]. Those aminoglycosides with 2-deoxystreptamine will bind to and stabilize a conformation of the A-site very similar to the “on” state, which allows the binding of non- cognate tRNAs to the A-site, resulting in misreading of mRNA and the false synthesis of proteins [38]. Although other classes of aminoglycosides may retain a distinct binding mode to the 30S subunit, their interactions with and effects on the A-site are highly conserved with 2-deoxystreptamine aminoglycosides [38]. For instance, spectinomycin (Fig 1.4), an aminocyclitol aminoglycoside, binds to the 30S subunit in a different location but very close to the A-site, where it can interfere with protein synthesis in a similar way.

Figure 1.4 Structures of representative aminoglycosides: 2-deoxystreptamine, streptomycin, kanamycin A, spectinomycin, neomycin C, gentamicin

The most prevalent mechanism of resistance to aminoglycoside comes from aminoglycoside modifying enzymes, including aminoglycoside acetyltransferases, phosphotransferases and nucleotidyltransferases. These enzymes can modify the amino groups (acetylation) or hydroxyl groups (phosphorylation, transfer of AMP group) of the aminoglycoside, leading to the inactivation of aminoglycosides [39]. Target modification or mutation is another source of resistance. Alteration in the binding sites or other parts of the ribosome which can impair the binding affinity will confer resistance of aminoglycosides. Bacterial strains with high-level aminoglycoside resistance in which the active site of the 16S rRNA has been modified by methyltransferases have been widely reported [40]. New aminoglycoside antibiotics with novel structures have been developed recently, including the recent FDA approval of plazomicin [41]. Plazomicin was approved to treat serious bacterial infections due to multidrug-resistant Enterobacteriaceae in 2018. Plazomicin preserves effective potency in the presence of various aminoglycoside- modifying enzymes, but it is not effective against bacteria with 16S rRNA methyltransferase [41]. 9

1.2.2.2 Macrolide antibiotics

Since the discovery of erythromycin (Fig 1.5), the macrolides have ranked as the third biggest family of antibiotics on the market [42]. From the viewpoint of structure, they belong to the polyketide class of natural products and contain a large lactone ring, as shown in Fig 1.5 [42]. Macrolides bind to the 50S subunits of the bacterial ribosome, thus inhibiting protein synthesis. In detail, they bind to a site on the 23S rRNA that is adjacent to the peptidyltransferase center within the ribosomal exit tunnel, thereby blocking the extension of the nascent peptide chain [43]. Resistance to macrolides can be caused by phosphorylation or glycosylation of the macrolide catalyzed by acquired bacterial rRNA enzymes [44]. Ribosomal modifications which cause reduced affinity with macrolides also lead to resistance [44]. For example, methylation of A2058 in the 23S rRNA renders bacteria resistant to macrolides [45]. The development of new macrolides antibiotics has continued. An erythromycin derivative compound with much higher binding affinity to the 50S subunit, telithromycin (Fig 1.5), was approved in 2001 [46]. However, severe adverse effects of telithromycin, including taste disorders, visual disturbances and liver damage [47], resulted in the withdrawal of its approval for use in simple infections such as sinusitis and bronchitis [48].

Figure 1.5 Structures of representative macrolides antibiotics: erythromycin and telithromycin

1.2.2.3 Tetracycline antibiotics

Tetracycline antibiotics have been used for decades as broad-spectrum antibiotics. As shown in Fig 1.6, the name of this class derives from the core scaffold of the molecule, which contains four fused hydrocarbon rings substituted with a variety of functional groups. Since the introduction of tetracycline into medical use in the 1950s, a number of tetracycline compounds have been used clinically [49]. In 2018, three tetracycline antibiotics were approved by FDA, omadacycline [50], sarecycline [51] and eravacycline [52]. The structure-activity relationships for the tetracyclines have been documented [49]. Tetracyclines bind to the 30S subunit and inhibit the binding of peptidyl-tRNA to the ribosome complex, but the exact binding site of tetracyclines remains controversial [53]. Resistance to tetracyclines can be mediated by modification of the tetracycline molecule or the modification of ribosomal rRNA or proteins, which interferes with binding [54]. However, the most prevalent resistance to tetracyclines results from efflux, which pumps tetracycline molecules out of the bacterial cell and leads to sub-therapeutic cytoplasmic concentrations of the tetracyclines [53].

Figure 1.6 Structures of representative tetracycline antibiotics

1.2.2.4 Oxazolidinone antibiotics

As a class of synthetic antibiotics, the oxazolidinones are heterocyclic molecules with

11 a nitrogen and oxygen in a five membered ring connected by a carbonyl group [55]. Linezolid (Fig 1.7) was the first antibiotic of the oxazolidinone class. As a drug of last resort, linezolid is used to treat serious infections caused by multidrug-resistant Gram- positive bacteria, including vancomycin-resistant Enterococci (VRE) and MRSA [56]. The mechanism for the oxazolidinone antibiotics can be understood by examining the action of linezolid, which prevents bacterial protein synthesis by binding to the 50S subunit. Linezolid can block the precise positioning of tRNA in the A-site of the peptidyltransferase center [57]. Mutations in ribosomal proteins and the 23S rRNA nucleotides that interact with linezolid are one of the major mechanisms of resistance. Modification of 23S rRNA by methyltransferase also causes resistance to linezolid [56]. Tedizolid (Fig 1.7) is another example of an oxazolidinone antibiotic, and it possesses enhanced potency as compared to linezolid [58]. In 2014, tedizolid was approved for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms, including MRSA [58]. Cross- resistance between tedizolid and linezolid exists, arising from mutations in 23S subunit rRNA or ribosomal proteins [58]. However, resistance to the oxazolidinone class in clinical settings has been very rare to date [59].

Figure 1.7 Structures of linezolid and tedizolid

1.2.3 Cell membrane antibiotics

Some polypeptide and lipopeptide molecules, which disrupt the bacterial cell membrane, are used as antibiotics in agriculture, in the food industry and as therapeutics [60-62]. Colistin (Fig 1.8), also named as polymyxin E, is the representative Gram-negative bacterial antibiotic of cyclic polypeptide class. Colistin competitively displaces Ca2+ and Mg2+ in the lipopolysaccharides of the outer membrane of Gram-negative bacteria [62], 12 thereby demonstrating a potent bactericidal effect. However, due to its common and serious nephrotoxicity and neurotoxicity side effects [63], colistin is used as a “last-line” therapy against multidrug-resistant and extensively drug-resistant Gram-negative bacterial infections when all other antibiotics are ineffective [64]. Resistance to colistin is rare in the hospital at this moment, yet some resistant strains caused by the change of bacterial outer membrane components have been reported [65, 66]. Resistance mediated by the MCR-1 mechanism has even been discussed in public media [67].

Figure 1.8 Structures of colistin and daptomycin

A lipopeptide consists of a cyclic peptide and lipid chain. A number of lipopeptides have been used as antiviral, antifungal and antibacterial reagents [60, 61]. Daptomycin (Fig 1.8) is the only antibiotic of the lipopeptide class in medical use till now [68]. Daptomycin has great potency against most clinically relevant Gram-positive organisms, including VRE and MRSA. Since its approval to treat complicated skin and skin structure infections in 2003, daptomycin has been widely used and listed as a “drug of last-resort” [68]. Currently, the exact mechanism of daptomycin is not fully understood [69]. It is known to insert into the cell membrane through the interaction with a bacterial membrane lipid, phosphatidylglycerol. The insertion proceeds in a Ca2+-dependent manner, followed by the aggregation of daptomycin in cell membrane, which causes rapid depolarization of the cell membrane and subsequent loss of cytoplasmic components, including amino acids, nucleotides, K+, Mg2+, ATP and so on [70]. Resistance to daptomycin is mainly mediated

13 by adaptive changes to the cell membrane [71].

1.2.4 Folate biosynthesis antibiotics

Folate is essential for cell growth and survival. The reduced form of folate, tetrahydrofolate, acts as a 1-carbon donor for the biosynthesis of bases in nucleic acids, such as purines and thymidine [72]. The biosynthesis of tetrahydrofolate can be simplified as follow: p-aminobenzoate (PABA) is incorporated into dihydropteroate by dihydropteroate synthase. Then the dihydropteroate is transformed into dihydrofolate, which is reduced to tetrahydrofolate by dihydrofolate reductase. Both dihydropteroate synthase and dihydrofolate reductase are antimicrobial targets [73]. Sulfonamides are a family of antifolate antibiotics, including sulfamethoxazole, sulfafurazole and sulfadoxine (Fig 1.9). They work as competitive inhibitors of dihydropteroate synthase, and compete with PABA for incorporation into dihydropteroate [74]. Trimethoprim (Fig 1.9), an inhibitor of dihydrofolate reductase, is used in combination with sulfamethoxazole. Synergistic antimicrobial activity results from their serial inhibition of bacterial folate biosynthesis [75]. Unfortunately, allergy to sulfonamide drugs is common among patients [76]. Sulfonamide and trimethoprim are widely used due to their low costs, resulting in high resistance in bacteria [77, 78]. The resistance can be initiated by mutational or regulational changes of target enzymes (dihydropteroate synthase, dihydrofolate reductase), and low permeability and efflux of drugs [77].

Figure 1.9 Structures of sulfadoxine, sulfamethoxazole, sulfafurazole and trimethoprim

1.2.5 DNA transcription antibiotics

As the first step of gene expression, DNA transcription is essential to cell survival. The key enzyme of DNA transcription, RNA polymerase, is a valid target for antibacterial reagents [79]. The rifamycin family, including rifampin (Fig 1.10), rifabutin, rifapentine, and rifaximin (Fig 1.10), are the most significant RNA polymerase antibiotics in the clinic. Rifampicin is a first-line drug for mycobacterial infections, especially tuberculosis [80]. Rifampicin binds to the prokaryotic RNA polymerase with much higher affinity than the orthologous mammalian enzyme [81]. The rifampicin binding site is in the β subunit of RNA polymerase and adjacent to (12 Å away from) the catalytic Mg2+ position of the enzyme [82]. The binding results in strong steric clashes with the growing the RNA chain and thus inhibits bacterial DNA transcription [82]. Resistance to the rifamycin family is commonly observed in clinical use and mainly comes from the mutations in the β subunit of RNA polymerase, which can reduce the binding affinity of the rifamycins [83].

Figure 1.10 Structures of rifampicin, rifaximin and fidaxomicin

Fidaxomicin (Fig 1.10) is a novel macrolide antibiotic that works as a bacterial RNA polymerase inhibitor. Fidaxomicin has a narrow spectrum and possesses potency against 15

Gram-positive infections, especially Clostridioides difficile infection [84]. With excellent activity and significantly fewer recurrences for C. difficile infection [85], fidaxomicin was approved by FDA in 2011. Different from the rifamycin family, fidaxomicin binds to RNA polymerase in the initiation stage of transcription. In detail, fidaxomicin binds to the complex of the DNA template and RNA polymerase, which prevents the opening of double-stranded DNA during the initiation stage of DNA transcription [86]. Consequently, cross resistance between fidaxomicin and the rifamycin family is not observed.

1.2.6 DNA replication antibiotics

DNA replication is a significant step for DNA-based genetic inheritance. Bacterial enzymes involved in DNA replication, including type II topoisomerases (DNA gyrase and topoisomerase IV), DNA primase, DNA helicase, DNA polymerase and DNA ligase, differ from mammalian analogous enzymes in sophisticated structural features or utilized cofactors, which makes them good targets for antimicrobial drug discovery [87]. Although no antibacterial reagents against DNA primase, DNA helicase, DNA polymerase or DNA ligase have yet been approved for clinical use, efforts to develop such types of antibiotics have been continual [88]. Inhibitors of type II topoisomerases are the only antibiotics obstructing DNA replication which are widely used in anti-infective medicine. ATPase inhibitors, quinolones, and novel bacterial topoisomerase inhibitors (NBTI) are major classes of topoisomerase-targeting antibiotics [89]. The latter two types will be discussed specifically in the following sections.

Figure 1.11 ATPase inhibitor: novobiocin

ATPase inhibitors can bind to the ATP-binding domain of the topoisomerase enzymes, which provides energy and is required for enzymatic function (details illustrated in

16 following section). Novobiocin (Fig 1.11) and other aminocoumarin antibiotics belong to the family of ATPase inhibitors [90]. Novobiocin was approved in the 1960s, but poor bioavailability, toxicity concerns, as well as resistance limited its clinical use [91]. In vivo studies about new ATPase inhibitors based on the structure of novobiocin have been documented [92, 93].

1.3 Quinolones as Antibiotics

Since the launch of first commercial quinolone, nalidixic acid (Fig 1.12), as an antibacterial drug in 1962 [94], the quinolone family has become one of the most economically successful antibiotic classes globally. Quinolone antibiotics show excellent bactericidal activity by selectively targeting bacterial type II topoisomerases over human topoisomerase II. With the inexpensive accessibility and good bioavailability, the quinolone antibiotics were widely used as first-line treatments for a variety of infections [95] but have lost popularity in recent years due to various adverse effects [96].

Based on the structural features and spectrum of antibacterial activity, the quinolone family can be classified into four generations [97]. Nalidixic acid is typical of the quinolone agents of the first generation, which are non-fluorinated drugs and possess only moderate Gram-negative activity [98]. All the second-, third- and fourth-generation quinolones are fluorinated compounds, so they are called fluoroquinolones. The second-generation quinolones have improved antimicrobial spectrum as compared to the first-generation, gaining extended Gram-negative activity, especially against aerobic microorganisms, but they possess more limited Gram-positive activity [97]. Ciprofloxacin is the prototypical second-generation quinolone and is the leading fluoroquinolone in sales [95]. Norfloxacin and lomefloxacin (Fig 1.12) are other examples for the second generation. The third- generation quinolones maintain the spectrum of second-generation quinolones and improve the Gram-positive coverage [97]. Levofloxacin and sparfloxacin (Fig 1.12) belong to the third generation. The fourth-generation quinolones show improved Gram-positive and anaerobic activity and have dual inhibition of both DNA gyrase and topoisomerase IV, which will be discussed later. Thus they possess a lower rate of resistance. [97] 17

Gatifloxacin, delafloxacin and moxifloxacin (Fig 1.12) are representative fluoroquinolones of the fourth generation.

Figure 1.12 Representative quinolones of first, second, third, and fourth generations

1.3.1. Mechanism of action for quinolones

As mentioned previously, the mechanism of action for quinolones is the inhibition of bacterial type II topoisomerase enzymes. Most bacteria have two type II topoisomerases, DNA gyrase and topoisomerase IV. DNA gyrase is the primary target of quinolones in most Gram-negative bacteria, while topoisomerase IV is preferentially targeted in many Gram-positive bacteria [99]. Consistently, the fourth-generation quinolones gain improved Gram-positive activity, and they retain dual inhibition against DNA gyrase and topoisomerase IV.

These two topoisomerase enzymes are able to modulate DNA topology, which is vital

18 for DNA replication [87]. Both enzymes make a double-stranded cut in one of the DNA double helices and reseal it after the passage of another strand through the gap. The primary function of DNA gyrase is to relax positive supercoils and introduce negative supercoils, while the main task of topoisomerase IV is to catalyze DNA decatenation. The two enzymes have substantially homologous amino acid sequences and have similar functional pathways. Both enzymes are A2B2 heterotetramers composed of two pairs of identical subunits, GyrA2GyrB2 for DNA gyrase and ParC2ParE2 for topoisomerase IV. GyrA is homologous to ParC while GyrB is homologous to ParE [100]. The GyrB and ParE subunits contain the ATPase domains. Herein, the mechanism of DNA gyrase is described in detail [101].

As shown in Fig 1.12, the DNA double strands can be divided into the gate segment (G-segment, red) and transport segment (T-segment, dark red). The two GyrB subunits form the N-gate of the complex and the two GyrA subunits form the C-gate. 1) The G- segment binds to the interface of GyrA and GyrB; 2) The N-gate opens and places the T- segment inside it; 3) Two ATP molecules bind to the ATP domains in the GyrB, which leads to the N-gate closing; 4) With the hydrolysis of one ATP molecule, the G-segment is cleaved by the active tyrosine in the GyrA subunits, forming a DNA-protein covalent intermediate. Then the T-segment passes through the cleaved G-segment; 5) With the hydrolysis of a second ATP molecule, the C-gate open and the N-gate closed, the T- segment is released from the complex. Meanwhile, the cleaved G-segment is resealed by the enzyme. In this process, the role of ATP hydrolysis is not fully understood, but it is clear that it serves as the energy source for enzymatic reactions [102], which rationalizes the topoisomerase inhibitory behavior of ATPase inhibitors.

Figure 1.13 Mechanism relaxation of supercoiling by DNA gyrase [101]

Quinolones inhibit the function of the type II topoisomerases by blocking the resealing of the cleaved DNA strand. Quinolones bind to the interface of DNA and the enzymes near the active tyrosine in GyrA, forming a ternary quinolone-DNA-enzyme complex, which can stabilize the cleaved DNA strand [103]. Several crystal structures of the ternary complex showed a similar binding mode and demonstrated that quinolones intercalate into the DNA and interact with the enzymes in the catalytic pocket [104-107]. In the example of a crystal structure of moxifloxacin in complex with Acinetobacter baumannii topoisomerase IV, a Mg2+ ion mediates the interaction between quinolone and the enzyme. The Mg2+ ion is coordinated with two oxygens from the quinolone and four water molecules, and two of the Mg2+ coordinating water molecules make hydrogen bonds with

ParC S84 and E88 [107]. Through the interaction with topoisomerases, quinolones block normal DNA replication and other bacterial activities. Incomplete DNA breaks are generated and accumulated, which are toxic to the cell. These DNA breaks can stimulate the bacterial SOS response and other DNA repair pathways [108], and the bactericidal activity of quinolones is enhanced as the DNA repair is incomplete [109]. Hence, the formation of DNA breaks and the subsequent bacterial response are associated with the death of bacterial cells.

1.3.2. Resistance to quinolones

Resistance to quinolones was discovered simultaneously with their introduction into medical use. The wide use of quinolones around the world led to a relatively high resistance rate [110]. The resistance can be initiated from three general mechanisms: mutations in the topoisomerase enzymes, mutations that reduce drug accumulation, and plasmid-mediated quinolone resistance.

Mutations in the topoisomerase enzymes which reduce the binding affinity account for the majority of quinolone resistance. Most of these resistant mutations are located in the amino terminal domains of GyrA or ParC, which are near the active site tyrosine [103]. Typically, serine substitutions take up more than 90% of the mutant pool in both laboratory and clinical isolates [111]. As mentioned above, ParC S84 and E88 contribute to the interaction of quinolones and the A. baumannii topoisomerase IV enzyme through a Mg2+ bridge. Similarly, mutations in the corresponding residues in DNA gyrase and topoisomerase IV of diverse bacterial species are most commonly involved in quinolone resistance [107], such as mutation in GyrA S83 and D87 of Escherichia coli [112]. Since current quinolones can inhibit both DNA gyrase and topoisomerase IV, dual-target mutations in both DNA gyrase and topoisomerase IV generally lead to much higher level of resistance than single-target mutations do [113]. Stepwise accumulation of mutations in both topoisomerase enzymes leads to elevated levels of quinolone resistance [114].

Efficacious cytoplasmic concentrations of the quinolones are required for activity, and intracellular concentrations are regulated by both influx and efflux of the drug molecules.

Increased efflux and reduced influx of quinolones can confer resistance [110]. In Gram- negative species, the outer membrane creates a barrier for the inward diffusion of hydrophilic drugs, and influx relies on protein channels called porins. Porin downregulation or modification of porin channels can interfere with drug influx and lead to low-level resistance to quinolones [115]. In contrast, the influx of quinolone is diffusion- mediated in Gram-positive bacteria, and it has not been reported to cause resistance. The elevated expression of efflux pumps has been shown to cause quinolone resistance in Gram-negative bacteria [116]. Active efflux mediated by transporters can also cause low- level resistance to quinolones in Gram-positive bacteria [114].

Plasmid-mediated quinolone resistance was first reported in the 1990s and has become an emerging clinical problem [117, 118]. Plasmids carrying quinolone resistance genes can be transmitted horizontally and vertically. Encoded by these plasmids, functional proteins can lead to resistance against quinolones through various pathways. For instance, they can bind to topoisomerase enzymes and inhibit quinolones from interacting with the DNA- enzyme complex [119]; they can catalyze the modification of the structure of drug molecules [120]; and the efflux of quinolones can be elevated by plasmid-mediated transporters, including OqxAB and QepA [121].

1.3.3. Adverse effect of quinolones

The potential toxicities and adverse effects of quinolones have gained much attention since the 1990s [122, 123]. Some quinolones have been withdrawn from the market because of serious side effect, including gatifloxacin (hypoglycemia), trovafloxacin (hepatotoxicity) and temafloxacin (haemolytic uremic syndrome) [124]. The adverse reactions to quinolones also include symptoms of the central nervous system (CNS), complaints of gastrointestinal tract, skin symptoms and acute anaphylactoid reactions [125]. Over recent years, FDA has issued several “black box” warnings about potential severe adverse effects of the fluoroquinolones, including increased risk of tendinitis and tendon rupture, myasthenia gravis, irreversible peripheral neuropathy, risks to mental health and low blood sugar adverse reactions [126]. Very recently, the use of fluoroquinolones was

22 reported to bring increased risk of aortic aneurysm in a hospital setting [127]. Hence, the benefits of quinolone therapies should be measured against their risks. The present day use of quinolone antibiotics in children is restricted to life-threatening infections only for specific indications when no alternative agent is effective [124].

In summary, quinolone antibiotics display outstanding bactericidal activity via blocking the catalytic site of bacterial type II topoisomerase enzymes. However, the high rate of resistance and potential severe adverse effects have limited the use of quinolones. New inhibitors of the topoisomerase enzymes are in demand, aiming to overcome current resistance and improve safety issues.

1.4 Novel Bacterial Topoisomerase Inhibitors

Addressing the emergence of ubiquitous resistance to all classes of antibiotics requires the discovery of new antibiotic classes with unprecedented mechanisms of action [2]. An ideal solution is to discover new antimicrobial targets, which is particularly challenging since most obvious antibacterial targets have already been utilized. One potential alternative approach is to develop new chemotypes that inhibit validated targets using a distinct binding mode from current antibiotics. In this vein, Novel Bacterial Type II Topoisomerase Inhibitors (NBTIs) represent a promising class from recent years.

1.4.1. Typical structures of NBTIs

Figure 1.14 Structures of representative NBTIs

The class of NBTIs can be traced to 1998, when RPR (later Aventis and then Novexel) identified the hit NBTI as dual inhibitors of DNA gyrase and topoisomerase IV though screening [128]. Soon after that, GSK disclosed the first patent of NBTIs in 1999 [129]. The recent development of NBTIs has been progressive and encouraging. For instance, gepotidacin (Fig 1.14), the most advanced NBTI in clinical trials, has successfully completed Phase 2 clinical trial for the treatment of skin infections and gonorrhea [130, 131]. NXL101 (Fig 1.14) underwent a Phase 1 clinical trial but was withdrawn due to severe QTc prolongation [132]. Some NBTIs afforded favorable preclinical properties, such as ACT-387042 (Fig 1.14) [133] and NBTI5463 (Fig 1.14) [134].

1.4.2. Mechanism of action for NBTIs

Similar to the quinolone class, NBTIs target bacterial type II topoisomerase enzymes, but they have been shown to have different behaviors and mode of action. Some NBTIs have been reported to stabilize enzyme-mediated single-stranded DNA breaks, while fluoroquinolones only induce double-stranded DNA breaks [135]. Moreover, NBTIs inhibit type II topoisomerase by stabilizing the complex of pre-cleaved DNA and enzyme, preventing the cleavage of DNA [105, 136], while quinolones will stabilize the cleaved DNA strand and block the reunion of cleaved DNA segments. Hence, NBTIs maintain 24 potent activity against quinolone-resistant strains [132-134, 137]. Resistance to NBTIs typically occurs from mutations in DNA gyrase. For example, strains resistant to the NBTI NXL101 commonly show alteration of D83 and M121 in S. aureus GyrA, while the most frequent mutations associated with quinolone resistance are to S84 [132]. The structural basis of this important difference will be discussed in the following section.

Figure 1.15 Typical structure of NBTIs represented by GSK299423 and NXL101

As displayed in Figure 1.15, the typical structure of an NBTI consists of three part [137]: a left-hand side heterocycle, a right-hand side heterocycle, and an aliphatic linker domain which contains a nitrogen atom connecting the two heterocycles. In 2010, the magnificent X-ray structure of the ternary complex of S. aureus DNA gyrase, the NBTI GSK299423 and a 20-bp DNA segment was published [105], elucidating the binding mode of NBTIs with DNA gyrase. As shown in Fig 1.16 [105], GSK299423 (yellow) binds in the middle between the two active Mg2+ (replaced by an artifact Mn2+ in the crystallization), which are essential for the cleavage-religation processing of G-segment DNA [138]. NBTIs bind to DNA gyrase at a site which is distinct from that of quinolones (represented by ciprofloxacin) but still close in space. The left-hand side of GSK299423, which is a cyanoquinolone group, intercalates between the two central base pairs of the 20- bp DNA segment, whereas ciprofloxacin binds to the DNA at the cleavage site between bases No. 8 and No. 9 of the 20-bp DNA segment. The oxathiolopyridine group (right- hand side) of GSK299423 occupies a non-catalytic hydrophobic pocket between the two GyrA subunits. The moiety sitting in the hydrophobic pocket can make van der Waals interactions with the residues A68, G72, M75 and M121. The methylene group of the 25 oxathiol ring in GSK299423 and the carbonyl oxygen of A68 in the enzyme are proposed to form an unusual C-H hydrogen-bond interaction. At the entrance of the hydrophobic pocket, the nitrogen atom in the linker moiety of the NBTIs typically interacts with D83 of DNA gyrase, consistent with the observation that mutations of D83 can result in resistant strains to NBTIs [132]. Moreover, mutations of M121, which is nearby in space to D83 and also part of the hydrophobic pocket, can also lead to resistance to NBTIs [132]. Sequence analysis shows that the hydrophobic pocket is highly conserved in DNA gyrase and topoisomerase IV, which sets a reasonable theoretical foundation for the feasible dual inhibition of both DNA gyrase and topoisomerase IV by NBTIs.

Figure 1.16 [105] a) 2.1 Å gyrase GyrB27-A56 complex with GSK299423 shown in stick: GSK299423 (yellow) half-way between the two Mn2+ ions (purple) at the active site. 3.35 Å ciprofloxacin structure in spacefilling presentation (grey carbons) with the two Mn2+ ions (black); b) the oxathiolopyridine ring of GSK299423 sits in the hydrophobic pocket of the enzyme.

1.4.3. Previous chemical efforts to develop NBTIs

Medicinal chemistry efforts directed at the NBTIs have focused largely on three issues: 1) dual inhibition of DNA gyrase and topoisomerase IV; 2) broad spectrum antibacterial activity, especially against strains that are resistant to other antibiotics; and 3) reduced inhibition of the human cardiac potassium channel hERG. The dual target inhibition against DNA gyrase and topoisomerase IV can reduce the propensity for resistant pathogens

26 arising from simple mutations in either one of the enzymes, and the conserved amino acid sequence between the two enzymes offers the chance to design dual-targeting NBTIs. Although most NBTIs target DNA gyrase more potently, at least in S. aureus, several efforts to develop dual targeting compounds have been reported [139]. As illustrated in the mechanism of action and broad spectrum for quinolone class, inhibition of bacterial topoisomerases is expected to be lethal to most Gram-positive and Gram-negative bacteria. Additionally, the unique binding mode of NBTIs as compared to the quinolones and the distinct mechanism of action as compared to the other antibiotic classes lead to potent activity, including against MRSA. Inhibition of the human cardiac potassium channel encoded by the ether à-go-go-related gene (hERG) can lead to severe cardiotoxicity in the form of QTc prolongation and the potentially fatal arrhythmia Torsades de pointes. The hERG channel is partially responsible for the electrical activity of the heart. The hERG channel can pump K+ ions out of cardiac myocytes, which mediates the rapid delayed rectifier current in the repolarization of cardiac action potential and helps maintain normal cardiac rhythm [140, 141]. Inhibition of hERG has been commonly observed in the class of NBTIs. Thus optimization to reduce hERG inhibition is another important objective for the development of NBTIs [141, 142].

A wide variety of DNA-binding domains, linker domains and enzyme-binding domains have been screened and optimized to develop NBTIs with improved properties in terms of dual inhibition, the antimicrobial potency and cardiac safety issues [143, 144]. Except for the nitrogen atom which interacts with D83, the linker part of NBTIs does not make key interactions in the DNA-NBTI-enzyme complex. Consequently, substantial medicinal chemical innovations have been applied in this region, aiming to maintain potent activity and improve pharmacokinetic or safety properties [145]. Previous notable NBTIs will be introduced briefly based on the structural feature of linker groups.

1.4.3.1 NBTIs with aminopiperidine linker

Utilizing an aminopiperidine group as the linker, the remarkable tool molecule GSK299423 provided specific structural insight into how NBTIs bind DNA and DNA gyrase [105]. GSK299423 retained potent in vitro antibacterial activity against a variety of 27

Gram-positive and Gram-negative pathogens, including quinolone-resistant strains of MRSA, and displayed moderate potency against Proteus mirabilis and Pseudomonas aeruginosa [105]. Several groups have also conducted further research on NBTIs with the aminopiperidine linker. By incorporation of a pyridodioxine ring as the enzyme-binding domain and addition of a hydroxyl group in the linker part, NBTI-3101 (Fig 1.17, NBTI- 31xx series were named in this chapter) showed good potency against Gram-positive organisms S. aureus and Streptococcus pneumoniae, and the Gram-negative organism Haemophilus influenzae [146]. NBTI-3101 demonstrated efficacy against S. pneumoniae in rat respiratory tract infection model, but hERG inhibition by NBTI-3101 was relatively high (IC50=5M in patch clamp assay) [146]. Aiming to reduce the hERG inhibition via lower lipophilicity (logD), the N-linked bicyclic [2,3-b]pyrazin moiety was adopted as a DNA-binding domain in NBTI-3102 and NBTI-3103 (Fig 1.17) by a group at AstraZeneca, with the same pyridodioxine enzyme-binding domain [147]. NBTI-3102 and NBTI-3103 retained broad-spectrum antimicrobial activity and obtained reduced hERG inhibition

(IC50=31 M and 19 M, respectively in the Ion Works patch-clamp assay [148]). Exploration of more analogs of NBTI-3102 by the same AstraZeneca group led to NBTI- 3104 and NBTI-3105 (Fig 1.17) [149]. NBTI-3104, employing a pyridooxazinone moiety as the enzyme-binding domain, maintained broad spectrum antibacterial activity, with especially attractive activity against the serious Gram-negative pathogen P. aeruginosa

(MIC90=4 μg/mL). However, the moderate hERG inhibition (IC50 =35 M in Ion Works assay) of NBTI-3104 still caused prolongation of the corrected QT (QTc) interval in a dog toxicology study [149]. NBTI-3104 was further used as the lead compound in the development of more promising NBTIs [150], which will be discussed in the section on aminocyclohexane-linked NBTIs (1.4.3.4).

In order to further decrease hERG inhibition via reduced pKa, NBTI-3105 introduced an electron-withdrawing fluoro-substituent in the aminopiperidine linker. This compound displayed strong activity against a MRSA strain in a neutropenic mouse thigh infection model and significantly decreased hERG inhibition (IC50=233 μM in Ion Works assay). Together with moderate clearance in the dog and an improved in vivo QT profile in a guinea pig model, NBTI-3105 was advanced into phase 1 clinical trials [149] but was discontinued 28 for undisclosed reasons [144].

Figure 1.17 NBTIs with aminopiperidine linker and bicyclic DNA-binding domain

Additional structure-activity relationship studies of the aminopiperidine-linked NBTIs lead to the discovery of tricyclic DNA-binding motifs. For example, GSK966587 (Fig 1.18) [151] exhibited broad spectrum activity against Gram-positive (S. aureus and S. pneumoniae) and Gram-negative (H. influenzae) bacteria. GSK966587 retained low hERG inhibition (IC50=239 μM in patchXpress assay [152]), moderate blood clearance in dog and monkey models, and efficacy against respiratory tract infections in a S. pneumoniae mouse model [151]. An X-ray crystallographic structure of GSK966587 complexed with the catalytic core of S. aureus gyrase and DNA was obtained, which demonstrated a similar binding mode to the GSK299423 complex [105], supporting a generalized structural basis for NBTI binding [151]. However, the progression of GSK966587 toward clinic trials was stopped due to the observation of hepatic portal tract lesions in a dog safety toxicology study across all dose levels [151].

Figure 1.18 NBTIs with aminopiperidine linker and tricyclic DNA-binding domains

The screening of a set of other tricyclic moieties as DNA-binding domains by the same GSK group identified another advantageous NBTI, GSK945237 (Fig 1.18) [153]. A 2.05 Å crystal structure of GSK945237 again showed a similar binding mode with GSK299423. GSK945237 retained broad spectrum activity against Gram-positive pathogens, S. aureus and S. pneumoniae, and the Gram-negative pathogen H. influenzae. GSK945237 possessed weak hERG inhibition (IC50 = 238 μM) and showed efficacy against fuoroquinolone- resistant S. pneumoniae in a rat respiratory infection model. GSK945237 also achieved promising in vitro ADME and in vivo pharmacokinetic attributes, which led to the initiation of a Phase 1 clinical trial [153]. Unfortunately, the progression of GSK945237 was discontinued because of specific ocular toxicity issues in pigmented rats and testicular effects in 3-month dog studies [153]. Gepotidacin (also named GSK2140944, Fig 1.19) also belongs to this series. As the most advanced NBTI till now, gepotidacin will be discussed in a separate section at the end of this chapter.

Figure 1.19 NBTIs with aminopiperidine linker and fused tricyclic DNA-binding domain

A series of NBTIs with a novel fused tricyclic DNA-binding domain (presented by

REDX05777, REDX06181 and REDX06213, Fig 1.19) were reported to have balanced inhibition of E. coli DNA gyrase and topoisomerase IV, with selectivity over human topoisomerase II [154]. For example, REDX05777 inhibits E. coli DNA gyrase/topoisomerase IV with IC50=0.29 and 0.25 μM, respectively, and REDX05777 has an IC50>100 μM of human topoisomerase II. This series of compounds also displayed low hERG inhibition: REDX06181 has a hERG IC50>100 μM; REDX05777 and REDX06213 have hERG IC50>33 μM. These REDX compounds were more potent against Gram- positive species. They also demonstrated good activity against some Gram-negative species, such as E. coli, A. baumannii, H. influenzae and Neisseria gonorrhoeae. However, their activity against Gram-negative strains showed no general superiority to ciprofloxacin [154].

1.4.3.2 NBTIs with tetrahydroindazole linker

NBTIs with a tetrahydroindazole linker moiety were identified via an SAR study of pyrazole-containing bicyclic linkers, and NBTI-3106 (Fig 1.20) was the lead compound [155]. NBTI-3106 displayed good potency against Gram-positive (S. aureus, S. pneumoniae) and Gram-negative strains (E. coli). Further optimization of the lead NBTI- 3106 resulted in several promising compounds, represented by NBTI-3107-NBTI-3110 (Fig 1.20) [156]. Replacement of the secondary amine by an amide group in the linker maintained Gram-positive activity (S. aureus and S. pneumoniae) but decreased Gram- negative activity. Against E. coli, the MIC of NBTI-3106 is 1 μg/mL while that of NBTI- 3107 is >128 μg/mL. Nevertheless, the addition of a halogen atom, such as chlorine or bromine on the thiazinone enzyme-binding moiety is able to recover Gram-negative activity of these amide-substituted tetrahydroindazole NBTIs. NBTI-3108, NBTI-3109, NBTI-3110 maintain promising potency against all the tested Gram-positive and Gram- negative strains, including S. pneumoniae, E. coli, and both fluoroquinolone-susceptible and -resistant MRSA strains [156]. Of note, these tetrahydroindazole NBTIs preserve dual enzymatic activity against S. pneumoniae DNA gyrase and topoisomerase IV [156]. For example, NBTI-3106 can inhibit S. pneumoniae DNA gyrase and topoisomerase IV with

IC50=0.25 μg/mL and 0.016 μg/mL, respectively [155]. 31

Figure 1.20 NBTIs with tetrahydroindazole linker

1.4.3.3 NBTIs with carboxypiperidine Linker

NXL101 (Fig 1.21) is a representative NBTI with a carboxypiperidine linker. The development of NXL101 was conducted by Novexel (acquired by AstraZeneca in 2009) and resulted in advancement to a Phase 1 clinical trial. Unfortunately, the development was discontinued due to QT interval prolongation in the Phase 1 clinical trial [132]. However, NXL101 possesses strong activity against Gram-positive bacteria, including fluoroquinolone- and methicillin-resistant strains. The in vivo efficacy of NXL101 was also demonstrated in murine infection models. Interestingly, the enzymatic inhibition of NXL101 is more potent for topoisomerase IV than DNA gyrase in E coli., whereas its inhibition of DNA gyrase is stronger than topoisomerase IV in S. aureus [132]. This inhibition profile in S. aureus is consistent with the observation that mutations of D83 and M121 in S. aureus gyrase caused resistance to NXL101 [132].

The replacement of the enzyme-binding thiophene of AVE6971 (the des-fluoro analog of NXL101, Fig 1.21) by a selenophene moiety led to NBTI-3111 (Fig 1.21) [157]. In vitro studies of NBTI-3111 displayed good methicillin-susceptible S. aureus and fluoroquinolone-resistant MRSA activity, low cytotoxicity in human cells, and high metabolic stability. NBTI-3111 and its diastereoisomer NBTI-3112 (Fig 1.21) have 32 moderate hERG inhibition with IC50=38 μM and 12 μM, respectively (patch clamp electrophysiology assay) [157].

Figure 1.21 NBTIs with carboxypiperidine linker

A series of carboxypiperidine NBTIs with a cyclobutylphenyl enzyme-binding moiety were reported, including NBTI-3113 and NBTI-3114 (Fig 1.21) [158, 159]. NBTI-3113 retains good whole cell activity against S. aureus but has weak activity against D83N mutant and M121K mutant S. aureus (both MIC≥64 μg/mL) due to inferior topoisomerase

IV inhibition (IC50=32.1 μM and 1.19 μM for topoisomerase IV and gyrase, respectively).

NBTI-3113 has moderate hERG inhibition with IC50=24.9 μM [159]. Addition of fluorine atoms afforded the lead compound of this series, NBTI-3114 [158]. NBTI-3114 achieved improved topoisomerase IV inhibition (IC50=5.47 μM), which translated into superior whole cell activity against gyrase D83N (MIC=3.4 μg/mL) or M121K (MIC=2 μg/mL) mutant S. aureus. NBTI-3114 demonstrated in vivo efficacy against fluoroquinolone- resistant S. aureus in a murine neutropenic thigh infection model and a systemic infection

33 model. The attenuated hERG inhibition of NBTI-3114 (IC50=85.9 μM) well explained the lack of prolongation or cardiac alternans in the anesthetized guinea pig [158].

1.4.3.4 NBTIs with aminocyclohexane linker

As mentioned previously, NBTI-3104 (Fig 1.14) possesses great activity against P. aeruginosa, but its hERG inhibition is problematic [149]. Starting from NBTI-3104, efforts to reduce hERG inhibition while preserving the Gram-negative activity have been made by the same group from AstraZeneca [150]. As a result, aminocyclohexane linked compounds, including NBTI-3115 and NBTI5463 (Fig 1.22), have been identified. The optimization was mainly focused on the reduction of logD in NBTI compounds based on their previous experience. Through the introduction of a second basic amine group, the logD of NBTI- 3115 was lower than that of NBTI-3104 (-0.36 vs 0.6, respectively). NBTI-3115 maintained excellent activity against P. aeruginosa (MIC90=4 μg/mL), including fluoroquinolone-resistant isolates. NBTI-3115 achieved significantly reduced hERG inhibition (IC50 >333 μM), and this compound also showed moderate clearance and high volume of distribution [150].

Figure 1.22 NBTIs with aminocyclohexane linker

With incorporation of fluorine on the quinoxalinone ring, NBTI5463 displayed broad spectrum activity as well as reduced hERG inhibition and superior pharmacokinetic properties as compared to NBTI-3115 [150]. In an MIC study against key Gram-negative bacterial pathogens, NBTI5463 showed promising activity against P. aeruginosa, E. coli and Citrobacter spp. (MIC90=2 μg/mL, 2 μg/mL and 4 μg/mL, respectively), but moderate activity against Proteus spp. and Stenotrophomonas maltophilia (MIC90=8 μg/mL for both), and weak activity against Klebsiella pneumoniae, Enterobacter spp. and A. baumannii

(MIC90=16 μg/mL, 16 μg/mL and 64 μg/mL, respectively) [160]. In vivo efficacy against

P. aeruginosa was demonstrated in a neutropenic mouse thigh infection model [150]. NBTI5463 was a potent dual-targeting inhibitor of both E. coli DNA gyrase and topoisomerase IV [160]. Consequently, a study of NBTI5463 resistance in E. coli was of interest [161]. Single mutations in E. coli gyrase such as D82G resulted in the loss of gyrase activity of NBTI5463, but the potent whole cell activity of NBTI5463 was preserved. Meanwhile, mutations in topoisomerase IV alone such as D79G only caused the loss of topoisomerase IV activity of NBTI5463 but maintained the whole cell potency. Only combined mutants in both DNA gyrase and topoisomerase IV were sufficient to confer resistance to NBTI5463. For instance, a strain with D82G in gyrase and D79G in topoisomerase IV resulted in a 128-fold increase in the MIC as compared with wild-type E. coli [161]. These results validated the value of dual-targeting enzymatic activity to mitigate resistance in the development of NBTIs. The reduced potential for QT prolongation of NBTI5463 was demonstrated in a study of monophasic action potential in the guinea pig model and a study of QT prolongation in the dog model [150]. Unfortunately, the preclinical safety profile of NBTI5463 was not sufficient for its further development [161].

1.4.3.5. NBTIs with tetrahydropyran linkers

A trans-disubstituted tetrahydropyran linker has been employed in a series of NBTIs from a group at Actelion [162-164, 138]. Preliminary exploration on this series developed the diol analogs as shown in NBTI-3116 and NBTI-3117 (Fig 1.23) [162]. NBTI-3116 and NBTI-3117 showed good potency against S. aureus and S. pneumoniae. NBTI-3117 was also identified as having exquisite activity against clinically relevant Gram-positive isolates, including fluoroquinolone-resistant S. aureus strains, MRSA and VRE. The antistaphylococcal activity was also confirmed in vivo in a neutropenic murine thigh infection model [162].

NBTI-3116 and NBTI-3117 possessed improved S. aureus topoisomerase IV inhibition (IC50= 0.5 μM and 8 μM for NBTI-3116 and NBTI-3117, respectively) compared with NXL101 (S. aureus topoisomerase IV IC50= 128 μM). As a result, NBTI-3116 resulted in 30-fold (at 4x MIC) and >100-fold (at 16x MIC) reductions in the rate of spontaneous 35 resistance in S. aureus as compared to NXL101. NBTI-3117 showed 40-fold (4x MIC) and 5-fold (16x MIC) reductions in the spontaneous resistance rate compared to NXL101 [162]. NBTI-3116 displayed unacceptable hERG K+ channel block (54% block at 10 μM), but hERG K+ channel block of NBTI-3117 (19% block at 10 μM) was decreased [162]. However, NBTI-3116 was still found to affect various cardiovascular parameters in anesthetized guinea pigs in subsequent studies [138]. These results led to the launch of further optimization studies on tetrahydropyran-linked NBTIs, aiming to obtain a more favorable cardiovascular safety profile.

Figure 1.23 NBTIs with tetrahydropyran linkers

With slight modification of the enzyme-binding domain and incorporation of a mono- hydroxylated linker, ACT-387042 and ACT-292706 (Fig 1.23) represented the fruits of this round of optimization [138]. ACT-387042 reduced the hERG K+ channel block (10% block at 10 μM, hERG inhibition IC50=128 μM [164]) and showed no effects on cardiovascular safety parameters in guinea pigs at free concentrations up to 8 μM. Meanwhile, ACT- 387042 retained strong antibacterial potency against many clinically significant Gram- positive pathogens. It displayed a low frequency of spontaneous resistance, superior

36 pharmacokinetic attributes in mice and good efficacy against S. aureus and S. pneumoniae strains in a neutropenic murine thigh infection model [138, 163]. Owing to this promising profile, a preclinical development program was initiated for ACT-387042 [138]. However, no clinical trials were ever reported.

Development of tetrahydropyran-linked NBTIs with expanded antibacterial spectrum covering some clinically problematic Gram-negative bacteria was conducted by the same group at Actelion [164]. Via the adoption of a primary amine, NBTIs with a dibasic tetrahydropyran linker were discovered, as represented by NBTI-3118 and NBTI-3119 (Fig 1.23) [164]. NBTI-3118 showed good activity against Gram-positive bacteria and several Gram-negative strains, including E. coli (MIC=0.5-2 μg/mL), Citrobacter freundii (MIC=1 μg/mL) and P. aeruginosa (MIC=1-2 μg/mL) [164]. NBTI-3119 presented broad spectrum whole cell activity against all selected Gram-positive strains (MIC≤2 μg/mL) and most Gram-negative pathogens (MIC=0.125-4 μg/mL), with the exception of Serratia marcescens (MIC=1 and 8 μg/mL). The in vivo efficacy of both compounds was demonstrated against relevant pathogens in neutropenic murine thigh infection models [164]. Both NBTI-3118 and NBTI-3119 maintained dual enzymatic activity against DNA gyrase and topoisomerase IV from Gram-negative organisms [164]. For example, NBTI-

3118 inhibited P. aeruginosa gyrase and topoisomerase IV with IC50=0.125 μM and 0.03 μM, respectively; NBTI-3119 inhibited E. coli gyrase and topoisomerase IV with

IC50=0.125 μM and 0.03 μM, respectively. Unfortunately, both compounds suffered from disadvantageous hERG inhibition. NBTI-3119 had a hERG IC50=5 μM, resulting in QT prolongation at a free concentration of 0.53 μM in the anesthetized guinea pig model.

NBTI-3118 inhibited hERG with IC50=52 μM and caused QT prolongation at a free concentration of 2.7 μM in this model [164]. These findings obstructed the further development of these compounds.

1.4.3.6. NBTIs with oxabicyclooctane linkers

Starting from AM8085 (Fig 1.24) [137], systematic SAR studies on NBTIs with an oxabicyclooctane linker have been disclosed by a group at Merck [137, 165-170]. AM8085

37 presented good whole cell activity against almost all tested Gram-positive and Gram- negative strains, including MRSA and VRE, but hERG inhibition by AM8085 was quite potent (IC50=0.6 μM) [137]. The addition of a hydroxyl group at C-2 of the linker gave rise to AM8191 and AM8192 (Fig 1.24), compounds with markedly diminished hERG activity

(IC50=18 μM for AM8191) [137]. The (S)-configured hydroxyl AM8191 was more potent than the (R) enantiomer AM8192. AM8191 retained good whole cell activity (MIC=0.015- 2 μg/mL) against most pathogens, with moderate activity (MIC=8 μg/mL) against P. aeruginosa. AM8191 showed balanced inhibition against gyrase and topoisomerase IV

(IC50=0.78 μM and 0.5 μM, respectively) from E. coli, but the S. aureus DNA gyrase inhibition was more potent than topoisomerase IV (IC50=1.02 μM vs 10.4 μM, respectively). The antibacterial potency of AM8191 was also demonstrated in vivo in murine models of S. aureus and E. coli infections. X-ray crystallographic studies revealed that the binding mode of AM8191 resembled that of previous NBTIs. The hydroxyl group in the linker had no direct interactions in the complex [137], which indicated the tolerability of the linker in NBTIs.

A series of SAR studies on DNA-binding domains [167, 168], enzyme-binding domains [169] and hydroxyl substitutions on the linker [170] of oxabicyclooctane linked NBTIs showed that most modifications were not tolerated in terms of antibacterial potency. Ether substitutions at C-2 of DNA-binding 1,5-naphthyridine analogs such as NTBI-3120 retained good S. aureus activity but failed to cover Enterococcus faecium and Gram- negative strains [167]. A tricyclic-1,5-naphthyridinone DNA-binding moiety was also employed, as shown in NBTI-3121, NBTI-3122 and NBTI-3123 (Fig 1.24). Through decreasing logD, significant attenuation of hERG activity was obtained for NBTI-3121

(IC50=174 μM) [165], NBTI-3122 (IC50=333 μM) [166] and NBTI-3123 (IC50=764 μM) [166]. In the case of these three compounds, incorporation of a fluorine in the enzyme- binding moiety or replacement of the pyridoxazinone by a pyridodioxane enzyme-binding moiety led to further improvements in the hERG profile. However, all the three compounds showed reduced whole cell activity, especially narrow Gram-negative spectrum [165, 166].

Figure 1.24 NBTIs with oxabicyclooctane linkers

An N-hydroxyethyl-3-chloro-1,5-naphthyridone moiety was used as a DNA-binding domain in AM8722 (Fig 1.24) [171]. AM-8722 displayed potent whole cell activity (MIC=0.125-2 μg/mL) across most Gram-positive and Gram-negative organisms, including clinical methicillin-resistant and vancomycin-resistant strains, but it possessed only moderate activity against P. aeruginosa (MIC=8 μg/mL). The in vivo efficacy of AM8722 was also demonstrated in a murine methicillin-susceptible S. aureus (MSSA) model of disseminated infection [171]. This compound showed balanced dual enzymatic inhibition in E. coli, but inhibited DNA gyrase more potently than topoisomerase IV

(IC50=0.83 μM vs 12.8 μM, respectively) in S. aureus. Consequently, high frequencies of spontaneous resistance in S. aureus were observed for AM8722 (>1.3x10-6 at 2x MIC, -7 6.7x10 at 4x MIC). AM8722 had an unfavorable hERG profile (IC50=3.8 μM), which is significantly interior to that of tricyclic-1,5-naphthyridinone analogs [171].

1.4.3.7. Gepotidacin

As mentioned previously, gepotidacin is currently the most advanced NBTI. It embodies a triazaacenaphthylene DNA-binding domain with an aminopiperidine linker, as shown in Fig 1.25. Gepotidacin has successfully completed Phase 2 clinical trials for the treatment of skin infections and uncomplicated urogenital gonorrhea [130, 131]. A recent crystal structure of S. aureus gyrase-gepotidacin-DNA ternary complex indicated similar binding mode with previous NBTIs [172]. This compound possesses robust in vitro activity against various bacterial pathogens, including MRSA, levofloxacin-resistant S. aureus and E. coli, and penicillin-nonsusceptible S. pneumoniae [173, 174]. In vivo efficacy against quinolone-resistant S. aureus and S. pneumoniae in a murine thigh infection model [175], MRSA in a murine lung infection model [176], and E. coli in a rat pyelonephritis model [177] have been demonstrated for gepotidacin. Gepotidacin has also demonstrated potent bactericidal activity against 25 N. gonorrhoeae isolates, including five ciprofloxacin-nonsusceptible strains [178], and has successfully completed a Phase 2 clinical trial to treat uncomplicated urogenital gonorrhea caused by N. gonorrhoeae [179].

In the Phase 2 clinical trial of gepotidacin for acute bacterial skin and skin structure infections, an MRSA isolate with high level resistance to gepotidacin (MIC>32 μg/ml) was discovered in one patient with clinical failure [130]. This isolate possessed a gyrase D83N mutation [130], which was consistent with the unpublished results from our group that gepotidacin inhibits DNA gyrase more potently than topoisomerase IV. Moreover, in the Phase 2 clinical trial for uncomplicated urogenital gonorrhea, gepotidacin was highly effective (>95%) for bacterial eradication of N. gonorrhoeae in adult participants, but high- level resistance to gepotidacin was observed for the two urogenital microbiological failures [131]. These resistant N. gonorrhoeae isolates contained the classical aspartate mutations

[131]. Although gepotidacin retains an extremely minimal hERG inhibition (IC50=1400 μM in the patch clamp electrophysiology assay), QTc prolongation was observed in phase 1 clinical trial [180]. Nevertheless, gepotidacin continues to progress toward the market, and it represents the promise of the NBTI class as antibiotics.

Figure 1.25 Structure of gepotidacin

In summary, NBTIs have demonstrated promising potential as new therapeutics for drug-resistant infections. Many NBTIs displayed excellent in vitro and in vivo antibacterial potency. Dual targeting inhibition of NBTIs against DNA gyrase and topoisomerase IV can effectively reduce the rate of resistant mutants, as shown in the cases of NBTI5463 [161] and NBTI-3116 [162]. In order to enhance cardiovascular safety, efforts to improve hERG inhibition have been made in the development of new NBTIs. Several NBTIs achieved favorable hERG profiles while retaining other promising preclinical properties, including ACT-387042 [138] and NBTI-3105 [149]. The successful completion of two Phase 2 clinical trials by gepotidacin is encouraging [130, 131] and has generated further confidence and enthusiasm for the development of the NBTI class as new clinical antibiotics.

1.5 Conclusions

In the history of modern medicine, highly diverse chemotypes have been utilized as antibiotics. Regardless of the mechanism of action involved, however, resistance has eventually developed after the clinical use of each antibiotic [1]. New antibiotic classes with unprecedented mechanisms of action are in urgent demand to postpone the day when multidrug-resistant bacteria (superbugs) conquer our world [2]. As one of the most successful antibiotic classes, the quinolone antibiotics have antibacterial function through the inhibition of bacterial type II topoisomerase enzymes, thereby preventing DNA replication in bacteria. Unfortunately, the high resistance frequency and potential severe adverse effects have begun to limit the clinical use of quinolones. The class of NBTIs have the same target as quinolone antibiotics. However, a distinct binding mode and mechanism

41 of action between NBTIs and quinolones, as demonstrated by pharmacological and X-ray crystallographic studies [105], have established the NBTIs as a new class of antibiotics. The recent development of NBTIs has been encouraging. Efforts to discover NBTIs with broad spectrum activity, dual target inhibition of DNA gyrase and topoisomerase IV, and favorable hERG profiles have resulted in several promising NBTIs. The discovery of additional NBTIs as new antibacterial therapies is worthy of further investment.

1.6 References

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pyridoxazinone substituted RHS analogs of oxabicyclooctane-linked 1,5- naphthyridinyl novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents (Part-6). Bioorg Med Chem Lett 2015, 25, 3636-3643. 170. Singh SB, Kaelin DE, Wu J, Miesel L, et al. C1–C2-linker substituted 1,5- naphthyridine analogues of oxabicyclooctane-linked NBTIs as broad-spectrum antibacterial agents (part 7). Med Chem Commun 2015, 6, 1773-1780. 171. Tan CM, Gill CJ, Wu J, Toussaint N, et al. In Vitro and In Vivo Characterization of the Novel Oxabicyclooctane-Linked Bacterial Topoisomerase Inhibitor AM-8722, a Selective, Potent Inhibitor of Bacterial DNA Gyrase. Antimicrob Agents Chemother 2016, 60, 4830-4839. 172. Gibson EG, Bax B, Chan PF, Osheroff N. Mechanistic and Structural Basis for the Actions of the Antibacterial Gepotidacin against Staphylococcus aureus Gyrase. ACS Infect Dis 2019, 5, 570-581. 173. Biedenbach DJ, Bouchillon SK, Hackel M, Miller LA, et al. In Vitro Activity of Gepotidacin, a Novel Triazaacenaphthylene Bacterial Topoisomerase Inhibitor, against a Broad Spectrum of Bacterial Pathogens. Antimicrob Agents Chemother, 2016, 60, 1918-1923. 174. Flamm RK, Farrell DJ, Rhomberg PR, Scangarella-Oman NE, Sader HS. Gepotidacin (GSK2140944) In Vitro Activity against Gram-Positive and Gram- Negative Bacteria. Antimicrob Agents Chemother 2017, 61, e00468-17. 175. Bulik CC, Okusanya ÓO, Lakota EA, Forrest A, et al. Pharmacokinetic- Pharmacodynamic Evaluation of Gepotidacin against Gram-Positive Organisms Using Data from Murine Infection Models. Antimicrob Agents Chemother 2017, 61, e00115-16. 176. So W, Crandon JL, Nicolau DP, Pharmacodynamic Profile of GSK2140944 against Methicillin-Resistant Staphylococcus aureus in a Murine Lung Infection Model. Antimicrob Agents Chemother 2015, 59, 4956-4961. 177. Hoover JL, Singley CM, Elefante P, Rittenhouse S. Efficacy of Human Exposures of Gepotidacin (GSK2140944) Against Escherichia coli in a Rat Pyelonephritis Model. Antimicrob Agents Chemother 2019, pii: AAC.00086-19. 178. Farrell DJ, Sader HS, Rhomberg PR, Scangarella-Oman NE, Flamm RK. In Vitro Activity of Gepotidacin (GSK2140944) against Neisseria gonorrhoeae. Antimicrob Agents Chemother 2017, 61, e02047-16. 179. Scangarella-Oman NE, Hossain M, Dixon PB, Ingraham K, et al. Microbiological Analysis from a Phase 2 Randomized Study in Adults Evaluating Single Oral Doses of Gepotidacin in the Treatment of Uncomplicated Urogenital Gonorrhea Caused by Neisseria gonorrhoeae. Antimicrob Agents Chemother 2018, 62, e01221-18. 180. Hossain M, Zhou M, Tiffany C, Dumont E, Darpo B. A Phase I, Randomized, Double-Blinded, Placebo- and Moxifloxacin-Controlled, Four-Period Crossover Study To Evaluate the Effect of Gepotidacin on Cardiac Conduction as Assessed by 12-Lead Electrocardiogram in Healthy Volunteers. Antimicrob Agents Chemother 2017, 61, e02385-16.

Chapter 2. Development of 5-amino-1,3-dioxane

Linked NBTIs with 6-methoxyquinoline DNA-

binding Moiety

2.1 Abstract

NBTIs represent a promising class of new antibiotics. However, the cardiac toxicity from the inhibition of hERG K+ channels have become one of the major challenges in the discovery of NBTIs. We proposed that the incorporation of a 5-amino-1,3-dioxane moiety as the linker domain would decrease hERG inhibition in new NBTIs. In this chapter, a series of new NBTIs with a 5-amino-1,3-dioxane linker and a 6-methoxyquinoline DNA- binding moiety were synthesized via a facile synthetic route. Diverse examples retained good whole cell activity. These NBTIs targeted DNA gyrase well but topoisomerase IV poorly. Meanwhile, these active compounds showed low inhibition of human homologous enzymes. The 5-amino-1,3-dioxane linker was also demonstrated to reduce hERG inhibition, substantiating our hypothesis and providing confidence for further optimization.

2.2 Introduction

As described in chapter one, NBTIs represent a prospective alternative therapy to treat bacterial infections. Recent progress in the discovery of new NBTIs has been promising, as several NBTIs have processed to clinical trials.[1] However, many of them failed due to a variety of issues. One of the major problems was the high propensity to inhibit the cardiac K+ channel encoded by the human ether-à-go-go-related gene (hERG), which is associated with cardiotoxicity [2]. For instance, viquidacin [3] was withdrawn because of QTc

53 prolongation observed in Phase I trials.[4] Gepotidacin (Figure 2.1) has recently shown promise in a Phase 2 trial for the treatment of acute bacterial skin and skin structure infections, including multidrug resistant strains.[5, 6] It is worth noting that significant (>10 ms) prolongation of the cardiac QT interval has been observed in clinical trials of gepotidacin even though it only minimally inhibits hERG (IC50 = 588 g /mL) [6], suggesting that some other cardiac ion channels may also contribute to the observed QT 2+ + + prolongation, including Cav1.2 Ca channel, Nav1.5 Na channel and KCND3 K channel [7]. Moreover, the blockage of hERG K+ channels and the corresponding potential for life- threatening arrhythmia has been documented in various non-antiarrhythmic drugs [2]. Addressing the safety issue of hERG inhibition has been a significant objective of modern medicinal chemistry programs, especially NBTI projects.[8]

DNA gyrase and topoisomerase IV are validated bacterial targets for the fluoroquinolone class of antibiotics as well as for the NBTIs [1, 9]. Both the binding mode and mechanism of inhibition of NBTIs are distinct from those of the fluoroquinolones [9- 11]. Consequently, typical NBTIs retain potent activity against fluoroquinolone-resistant strains [12]. As the inhibition of DNA gyrase is superior to that of topoisomerase IV for many documented NBTIs [13], resistance to NBTIs has frequently been observed from single amino acid substitutions in DNA gyrase [14, 15], including D83N, D83G, and M121K (all encoded by mutations in GyrA). Balanced dual-target inhibition for NBTIs appears to be an appropriate answer to lower the frequency of resistance, especially via the

54 improvement of topoisomerase IV activity [1].

Figure 2.1 Structures of AM-8085, ACT-387042, NBTI-5463, gepotidacin and GSK966587

As illustrated in Chapter 1 and shown in the structure of GSK299423 (Figure 2.2) [10], the typical NBTI pharmacophore consists of three parts: a “left-hand side” (LHS) domain which interacts with the DNA substrate, a “right-hand side” (RHS) domain which binds to the topoisomerase enzyme, and a linker domain. Except for the hydrogen bond (or ionic interaction if protonated) between the secondary amine and D83 of DNA gyrase (S. aureus numbering) [10], the linker of NBTIs is not involved in any significant interaction with either DNA or the enzyme. Consequently, substantial medicinal chemistry efforts have been made to tune physiochemical properties and achieve structural innovation through modification of the linker [1]. Examples (Figure 2.1) include the oxabicyclooctane linker in AM-8085 [16-18], tetrahydropyran linker in ACT-387042 [19-21], aminocyclohexane linker in NBTI-5463 [22], and the aminopiperidine linker in GSK966587 [23] and gepotidacin [9]. Some of these linkers have required complex synthetic routes for their preparation. For instance, the key intermediate of tetrahydropyran-linked NBTIs was obtained by an 8-step synthetic route [19, 24]. The oxabicyclooctane linker was another

55 extreme example, requiring 14 steps to construct the linker intermediate [16]. Thus, chemical feasibility represents another significant challenge and should also be taken into account in the design of new NBTI linkers.

Figure 2.2 Typical NBTI structure: GSK299423

In this work, a 5-amino-1,3-dioxane moiety was selected and designed as an innovative linker in a series of new NBTIs (Figure 2.3, Compound 9 as representative). Attractive features included the potential for diminished hERG inhibition and synthetic convenience, which would facilitate our efforts to improve topoisomerase IV inhibition. In previously reported NBTIs, substitution with an electron-withdrawing group in the linker moiety resulted in less potent hERG inhibition.[25, 26] Moreover, in the case of a selective PAK1 inhibitor G-5555 (Figure 2.3), the use of a 5-amino-1,3-dioxane moiety as a more polar and less basic replacement for piperidine conferred several benefits, including improved pharmacokinetics and decreased hERG inhibition.[27] Inspired by the above results, the 5- amino-1,3-dioxane linker was proposed as a means to reduce hERG inhibition. As shown in the methods section, the synthetic pathway to install a 5-amino-1,3-dioxane linker in NBTIs was extremely facile, enabling more convenient development of structure-activity relationships (SAR) for both the DNA-binding moiety and the enzyme-binding moiety. As a result, optimization of topoisomerase IV activity for our new NBTIs could be conducted more easily, aiming to achieve dual-target inhibition.

N Cl

O N O N H O N N N O O H O O

O NH2 N G-5555 Compound 9 Figure 2.3 Selective PAK1 inhibitor G-5555 and our representative NBTI, Compound 9

In order to evaluate the hypothesis of our design, a series of NBTIs with a 5-amino- 1,3-dioxane linker was prepared. On account of the ready accessibility of the 6- methoxyquinoline moiety, a library of different enzyme-binding RHS moieties with a 5- amino-1,3-dioxane-linked 6-methoxyquinoline DNA-binding component was established. The identification of enzyme-binding moieties with a favorable topoisomerase IV targeting domain was a principal objective. Anti-staphylococcal activity for these new NBTIs was tested. Additionally, dual-target inhibition (DNA gyrase and topoisomerase IV), hERG inhibition and preliminary human safety issues for selected compounds were also evaluated.

2.3 Results and Discussion

2.3.1 Synthesis

2.3.1.1 Synthesis of NBTIs possessing a 6-methoxyquinoline DNA-binding moiety

Scheme 2.1 Synthesis of NBTIs possessing a 6-methoxyquinoline DNA-binding moiety. Reaction conditions: i. DMF, PBr3, 95%; ii. toluene, 105 °C, 91%; iii. acrolein, CuSO4, p-TsOH (cat.), DCM, 76%; iv. a) 9-BBN, THF, 30 min. b) Compound 2, Pd(PPh3)2Cl2, Cs2CO3, THF, 54%; v. HO(CH2)2NH2, EtOAc, reflux, 75%; vi. NaBH3CN, ZnCl2, MeOH.

The synthesis of new dioxane-linked NBTI compounds containing a 6- methoxyquinoline DNA-binding moiety is depicted in Scheme 2.1. Quinoline intermediate 2 was synthesized via bromination of commercially available 4-hydroxy-6- methoxyquinoline (1) with PBr3 and was isolated in high purity (>95%) by precipitation and filtration. Serinol (3) was protected by phthalic anhydride (4) to give the phthalimide

58 derivative (5) in good yield (91%) and purity (95%). Catalyzed by p-toluenesulfonic acid, the condensation of diol 5 with acrolein proceeded to dioxane 6. Hydroboration of dioxane 6 with 9-BBN and subsequent Suzuki-Miyaura reaction of the resulting intermediate with quinoline bromide 2 afforded the coupled intermediate 7. Deprotection of the phthalimide group with ethanolamine in refluxing ethyl acetate [28] yielded intermediate 8. Finally, reductive amination between the primary amine 8 and a series of aldehydes gave rise to compounds 9-57.

Scheme 2.2 Structure of the library of compounds possessing a 6-methoxyquinoline moiety and a 5-amino- 1,3-dioxane linker. 60

Using a dioxane-linked 6-methoxyquinoline DNA-binding moiety, a library of analogs with different enzyme-binding moieties was prepared to study the structure-activity relationships (see Scheme 2.2). Among them, the last synthetic step of compounds 21-23, 25-27, 31, 32, 39, 42 was completed by Dr. Antony Okumu [29]. Compounds 56 and 57 were synthesized by Lovette Azap [29].

2.3.1.2 Synthesis of 3,4-dihydro-2H-pyrano[2,3-c]pyridine-6-carbaldehyde

OH O OH O OH O

i O OH O OPMB ii HN OPMB iii 58 59 60

OAc OH OAc O OAc OTf OH

OPMB iv N vi HN N OPMB v OPMB 61 62 63

OAc OAc OH OH N vii N viiiN ix OH O O 64 65 66

N O 67 Scheme 2.3 Synthesis of 3,4-dihydro-2H-pyrano[2,3-c]pyridine-6-carbaldehyde. Reaction conditions: i. PMBCl, DMF, K2CO3, 0 °C-85 °C, 79%; ii. ammonia, EtOH, 44 °C, 52%; iii. AcCl, pyridine, 0 °C to room temp. to 85 °C, 74%; iv. Tf2O, Et3N, DCM, 76%; v. propargyl alcohol, PdCl2(PPh3)2 (cat.), CuI2 (cat.), Et3N, MeCN, 94%; vi. Pd/C, H2, EtOH, 71%; vii. DIAD, PPh3, THF, used directly; viii. NaOH solution, 78%; ix. Dess–Martin periodinane, DCM, 88%.

3,4-dihydro-2H-pyrano[2,3-c]pyridine-6-carbaldehyde (compound 67) was synthesized by a minor modification of the documented method [30] in order to make compound 11. The synthetic pathway is shown in Scheme 2.3. Starting from kojic acid (58), the more acidic 5-hydroxyl group was deprotonated and protected by reaction with p-methoxybenzyl chloride (PMBCl), which gave compound 59. Reaction of compound 59 with ammonia afforded 4-pyridone 60. Acyl protection of the benzylic hydroxyl group produced compound 61. Subsequently, the trifluoromethanesulfonate intermediate (62) was obtained 61 by the addition of trifluoromethanesulfonic anhydride. Sonogashira coupling [31] between compound 62 and propargyl alcohol led to compound 63. Hydrogenation using Pd/C effected the clean reduction of the alkyne group and the concomitant cleavage of the PMB group, resulting in diol 64. The cyclization of diol 64 under Mitsunobu conditions afforded compound 65. Finally, hydrolysis of the acetate using an aqueous NaOH solution and subsequent Dess-Martin oxidation yielded compound 67.

2.3.1.3 Synthesis of 3-oxo-3,4-dihydro-2H-pyrido[3,2-b][1,4]thiazine-6- carbaldehyde

Scheme 2.4 Synthesis of 3-oxo-3,4-dihydro-2H-pyrido[3,2-b][1,4]thiazine-6- carbaldehyde. Reaction conditions: i. NBS, chloroform, 19%; ii. a) ethyl 2-mercaptoacetate, NaH, DMF, 0 °C. b) Compound 69, DMF, 37%; iii. NaOH solution, dioxane, 71%; iv. isobutyl chloroformate, Et3N, THF, -10 °C, used directly; v. NaBH4, THF/H2O, 64% (two steps); vi. Dess–Martin periodinane, DCM, 77%. 3-oxo-3,4-dihydro-2H-pyrido[3,2-b][1,4]thiazine-6- carbaldehyde (74) is the enzyme- binding motif of compound 12 and was synthesized by a minor modification of the route described by Miller [32]. The synthetic pathway is shown in Scheme 2.4. Bromination of the commercially available methyl 6-aminopicolinate (68) with N-bromosuccinimide (NBS) gave compound 69. Cyclization with ethyl 2-mercaptoacetate constructed the thiazine intermediate 70. Saponification of the methyl ester of compound 70 by aqueous NaOH produced the carboxylic acid intermediate 71. The carboxylic acid group in compound 71 was transformed into an isobutyl-acyl carbonate (72) with isobutyl chloroformate. The isobutyl-acyl carbonate of compound 72 was reduced directly by 62

NaBH4 to give alcohol 73. At last, Dess-Martin oxidation of the primary alcohol resulted in aldehyde 74.

2.3.1.4 Synthesis of 3-oxo-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazine-6- carbaldehyde

Scheme 2.5 Synthesis of 3-oxo-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazine-6- carbaldehyde. Reaction conditions: i. Br2, MeONa, MeOH, 0 °C, used directly; ii. ethyl bromoacetate, K2CO3, acetone, reflux, used directly; iii. Fe, AcOH, 90 °C, 16% (three steps); iv. phenylvinylboronic acid, K2CO3, Pd(PPh3)4, dioxane/H2O, 100 °C, 42%; v. OsO4, NaIO4, dioxane/H2O, 50%.

3-oxo-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazine-6- carbaldehyde (compound 80) was the key intermediate to prepare compound 13 and was synthesized following the reported method [16]. As shown in Scheme 2.5, compound 76 was obtained from the bromination of commercially available 3-hydroxy-2-nitropyridine (compound 75). The alkylation of compound 76 with ethyl bromoacetate then gave compound 77. The reduction of the nitro group with iron and cyclization of the oxazine ring were completed simultaneously in refluxing acetic acid. Suzuki coupling between bromide 78 and phenylvinylboronic acid led to compound 79. Finally, Lemieux-Johnson oxidation of the alkene afforded 3-oxo-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazine-6- carbaldehyde (80).

2.3.1.5 Synthesis of vinyl 6-methoxyquinoline moiety series

Scheme 2.6 Synthesis of vinyl compounds (86 and 87) possessing a 6-methoxyquinoline moiety. Reaction conditions: i. propargyl alcohol, CuI2, PdCl2(PPh3)2, Et3N, CH3CN, 50 °C, 76%; ii. Pd/C, H2, MeOH, 95%; iii. Dess-Martin periodinane, DCM, 61%; iv. p-TsOH (cat.), toluene, 110 °C, 59%; v. HO(CH2)2NH2, EtOAc, reflux, 73%; vi. NaBH3CN, ZnCl2, MeOH.

Two dioxane-linked vinyl compounds with a 6-methoxyquinoline moiety were synthesized to test the effect of a vinyl group in the linker of NBTIs, which was studied in previous NBTIs [19]. The synthetic pathway is straightforward (Scheme 2.6). Sonogashira coupling between bromide 2 and propargyl alcohol resulted in compound 81. The alkene group of compound 81 was reduced to an alkane (compound 82) via hydrogenation using Pd/C. An unusual Dess-Martin oxidation led to unsaturated aldehyde 83 with a (Z)-alkene,

64 which was identified by the coupling constant of the vicinal alkenyl protons (11.6 in aldehyde 83,). A similar oxidation by 2-iodoxybenzoic acid was reported before [33], and the driving force of the unusual oxidation is proposed to be formation of the conjugated system in unsaturated aldehyde 83. Condensation of aldehyde 83 and compound 5 produced the dioxane intermediate 84 with a (E)-alkene (coupling constant of the alkene protons is 15.9-16.0 in compound 84-87). The phthalimide group in intermediate 84 was then cleaved to release the primary amine 85. Finally, reductive amination of amine 85 with the appropriate aldehydes yielded the target compounds (86 and 87).

2.3.1.6 Synthesis of 6-methoxyquinoline analogs with a cyclohexane linker

Scheme 2.7 Synthesis of 6-methoxyquinoline analogs with a cyclohexane linker. Reaction conditions: i. a) 9-BBN, THF, 4 hrs. b) Compound 2, Pd(PPh3)4, K3PO4, EtOH/H2O, reflux, 70%; ii. trifluoroacetic acid, DCM, used directly; iii. NaBH3CN, ZnCl2, MeOH.

Four compounds possessing a cyclohexane linker were prepared as control compounds for comparison of hERG inhibition with the corresponding dioxane-linked analogs. The synthesis is straightforward as displayed in Scheme 9. Suzuki coupling between compound 88 and bromide 2 afforded compound 89 (carried out by Dr. Mark Mitton-Fry) [29]. The tert-butyloxycarbonyl group was then deprotected with trifluoroacetic acid to give the primary amine in compound 90, followed by reductive amination under our standard

65 conditions to yield final compounds 91-94. Compound 92 and compound 94 were synthesized by Dr. Antony Okumu [29].

2.3.1.7 Synthesis of 6-methoxyquinoline analogs with a piperidine linker

Scheme 2.8 Synthesis of 6-methoxyquinoline analogs with a piperidine linker. Reaction conditions: i. Pd(PPh3)4, K3PO4, DME/H2O, 70 °C, 89%; ii. chloroform, reflux, 65%; iii. trifluoroacetic acid, DCM, used directly; iv. NaBH3CN, ZnCl2, MeOH.

Analogously to the cyclohexane-linked compounds, four 6-methoxyquinoline analogs with a piperidine linker were synthesized. As shown in Scheme 2.8: compound 96 was obtained via a Suzuki coupling between bromide 2 and the commercially available vinylboronic anhydride pyridine complex 95. The heterogeneous mixture of compound 96 and commercial tert-butyl piperidin-4-ylcarbamate 97 was heated to reflux to give the coupling product (compound 98). The first two steps were conducted by Dr. Mitton-Fry [29]. Deprotection of the tert-butyloxycarbonyl group with trifluoroacetic acid afforded the primary amine intermediate 99, which was utilized to make final compounds 100-103 via reductive amination under our standard conditions. Compounds 101 and 103 were prepared by Dr. Antony Okumu [29].

2.3.2 Biological evaluation of NBTIs possessing a 5-amino-1,3-dioxane linker

2.3.2.1 Minimum inhibitory concentrations

Like other NBTIs, these newly synthesized compounds are expected to work as efficient antibacterial agents [10]. All of the new compounds were assayed in minimal inhibitory concentration (MIC) experiments to test the whole cell potency. For the library of 6-methoxyquinoline analogs with the 5-amino-1,3 dioxane linkers, MICs were determined to help understand the structure-activity relationships (SAR) of the enzyme- binding domain. The vinyl, cyclohexane and piperidine linked analogs were also tested to study the effect of those linkers on activity. These MIC assays were determined according to Clinical and Laboratory Standards Institute (CLSI) guidelines [34] using the S. aureus reference strain ATCC 29213 by our collaborator Sheri Dellos-Nolan in the laboratory of Professor Dan Wozniak at The Ohio State University [29]. Ciprofloxacin was used as positive control in each assay.

The MIC values for the library of 1,3-dioxane-linked 6-methoxyquinoline analogs are presented in Table 2.1. To our delight, nearly all of the compounds bearing the bicyclic enzyme-binding moieties (compound 9-17, 19, 55) possessed whole cell activity (MIC50 ≤

2 g/mL) comparable to ciprofloxacin (MIC50 = 0.125-0.5 g/mL), with the exception of compounds 18 and 54. For the 1-substituted naphthalene 18, its only structural difference from the 2-substituted naphthalene 17 is the orientation of enzyme-binding moiety (naphthyl group), but the MIC value of compound 17 was 1 g /mL while that of 18 was >8 g/mL, illustrating the significance of binding mode in retaining activity. The poor antibacterial activity of compound 54 also provided additional evidence for the importance of orientation. From the distinctive activity of naphthyl derivative 18 (>8 g/mL) and acenaphthylene derivative 19 (0.5 g/mL), we can see that the whole cell activity could be promoted by a bulky group which occupies the full depth of the hydrophobic pocket, which was also verified by the SAR of monocyclic derivatives described below.

The activity of monocyclic aromatic derivatives was highly dependent on the presence

67 and position of substitution. Non-substituted monocyclic derivatives, including phenyl ring (21), pyridyl ring (22, 23) and thiophenyl ring (53), did not show good antibacterial activity. Mono-substituted monocyclic derivatives retained activity dependent on the substituted site: neither the 3-substituted (26, 37-39, 42, 52) nor the 2-substituted (40, 41, 43) derivatives conferred potent activity, whereas many 4-substituted monocyclic analogs (25, 27-30, 33-36) were discovered with moderate activity (1-4 g/mL). Exceptions were compounds 31, 32 (>64 g/mL) and 51 (32 g/mL). It was hypothesized that the fluorine substitution in compound 31 was not suitable to take up the hydrophobic pocket in DNA gyrase due to its polarity and size, neither was the cyanide substitution in compound 32. For the di-substituted monocyclic derivatives, most of them (44, 46-50) were devoid of whole cell potency, but the 3,4-dichloro derivative 20 (≤ 0.25-1 g/mL) and 3-fluoro-4- chloro derivative 45 (2 g/mL) possessed potent activity. The non-aromatic cyclohexyl analog 56 had poor activity (8 g/mL) that was somewhat improved by incorporation of a 4-methyl substituent as in compound 57 (2 g /mL). Overall, it was observed that 4- substitution in the monocyclic derivatives improved the whole cell activity, which could be rationalized in accordance with the case of compounds 18 and 19, in other words that an appropriate substituent at the 4-position in the enzyme-binding domain of NBTIs could be an effective means to occupy the hydrophobic pocket in DNA gyrase.

Additionally, the X-ray crystal structure of GSK299423 [10] suggested an unusual hydrogen bond between the methylene of the oxathiolopyridine ring and the backbone carbonyl of A68 of DNA gyrase. We hypothesized that a similar novel hydrogen bond could be achieved by either a difluoromethoxy (35) or a difluoromethyl (36) substituent [35]. To our disappointment, neither of them proved superior to the non-fluorinated (27 and 29) or fully fluorinated (28 and 30) comparators.

To summarize the 6-methoxyquinoline library, bicyclic moieties appeared to serve as better enzyme-binding components than monocyclic moieties in the construction of new NBTIs, although monocyclic moieties with lipophilic 4-substitution also showed good potency.

Table 2.1. Minimal inhibitory concentrations (MICs) for 1,3-dioxane linked 6-methoxyquinolines

Compound S. aureus Compound S. aureus Compound S. aureus (MSSA) (MSSA) (MSSA) ATCC 29213 ATCC 29213 ATCC 29213 MIC MIC(g/mL) MIC(g/mL (g/mL)a,b a,b )a,b

9 0.25-1 26 32 43 >64

10 1-2 27 1 44 8

11 1 28 1 45 2

12 0.5 29 4 46 64

13 0.5 30 2 47 16

14 1-2 31 >64 48 64

15 1 32 >64 49 >64

16 2 33 2 50 64

17 1 34 4 51 32

18 >8 35 4 52 >64

19 0.5 36 2 53 >64

20 ≤0.25-1 37 64 54 >8

21 >64 38 16 55 1

22 >64 39 16 56 8

23 8 40 >64 57 2

24 8 41 >64 ciprofloxacin 0.125-0.5

25 1 42 32 aAssays were conducted according to CLSI guidelines (n=3, minimum). bRanges of observed values provided where appropriate.

Within this series, a vinyl group was incorporated into the linker domain to seek improved whole cell activity, but it offered no advantages as shown in Table 2.2. Neither 70 compound 86 (1-2 g/mL) nor 87 (0.5 g/mL) showed any advantage over their respective saturated analogs (9 and 20) against the S. aureus ATCC strain.

In order to study the effect of the 1,3-dioxane linker on hERG inhibition, cyclohexane- linked analogs 91-94 and piperidine-linked analogs 100-103 were prepared as structure- matched control compounds. The whole cell activity of cyclohexane-linked analogs 91-94 and piperidine-linked analogs 100-103 were tested first. The MIC values of compounds 91-94 ranged from 0.25-0.5 g/mL, and those of compounds 100-103 ranged from 0.125- 0.25 g/mL. In terms of antibacterial activity, the cyclohexane-linked and piperidine- linked compounds thus behaved similarly to their corresponding dioxane-linked analogs.

Table 2.2 MICs for 6-methoxyquinoline series with vinyl, cyclohexane, piperidine linker

R R NH N NH R= H O O N N O Cl O O N O O O Cl N N 91, 100 92, 101 93, 102 94, 103 91-94 100-103

O Cl

HN O HN Cl

O O O O

O O

N N 86 87 Compound S. aureus (MSSA) Compound S. aureus (MSSA) ATCC 29213 ATCC 29213 MIC (g/mL)a,b MIC(g/mL)a,b

86 1-2 94 0.5

87 0.25-0.5 100 0.125

91 0.5 101 0.25

92 0.25 102 0.125

93 0.25 103 0.125 aAssays were conducted according to CLSI guidelines (n=3, minimum). bRanges of observed values provided where appropriate.

Since the binding mode of typical NBTIs differs from that of fluoroquinolones [10], it was hypothesized that these new potent compounds would be active against fluoroquinolone-resistant cell lines. To test this point, new compounds with potent whole cell activity (typically ≤ 2 g /mL) against the ATCC 29213 strain were assayed for MICs using a USA 300 strain of MRSA [36] (Table 2.3). Vancomycin was used as positive control against USA 300. As displayed in Table 2.3, susceptibility to most of the selected compounds was similar between the ATCC and fluoroquinolone-resistant MRSA strain. In contrast, the MIC value of ciprofloxacin increased by 64-fold. Moreover, some of the selected compounds behaved as well as vancomycin against the MRSA USA 300 strain, illustrating a significant advantage of NBTIs, i.e. the retention of potency against clinically relevant fluoroquinolone-resistant strains.

Table 2.3 MICs for selected compounds in MSSA and MRSA cellular inhibitory experiments Compound S. aureus S. aureus Compound S. aureus S. aureus (MSSA) (MRSA) (MSSA) (MRSA) ATCC 29213 USA 300 ATCC 29213 USA 300 MIC(g/mL MIC MIC(g/mL MIC )a,b (g/mL)a,b )a,b (g/mL)a,b

9 0.25-1 0.5 27 1 1

10 1-2 2 28 1 2

11 1 2 30 2 4

12 0.5 0.5 33 2 4

13 0.5 1 36 2 4

14 1-2 1 45 2 1

15 1 2 55 1 2

16 2 4 56 8 8

17 1 2 57 2 2

18 >8 >8 86 1-2 1

19 0.5 1 87 0.25-0.5 0.5

20 ≤0.25-1 0.5 ciprofloxacin 0.125-0.5 16-32

25 1 4 vancomycin NT 1-2 aAssays were conducted according to CLSI guidelines (n=3, minimum). bRanges of observed values provided where appropriate.

2.3.2.2 Targeted enzyme inhibition

Based on our objective of achieving dual-targeting activity against both DNA gyrase and topoisomerase IV so as to reduce the rate of resistance, biochemical assays were applied on selected potent compounds to measure the inhibition of both enzymes. As shown in Table 2.4, compared with the positive control ciprofloxacin, almost all of the chosen compounds presented very potent inhibition of DNA gyrase, while most of them were inadequate to suppress the function of topoisomerase IV. For each compound, the inhibition of DNA gyrase was >100-fold that of topoisomerase IV. These results suggested that the anti-staphylococcal activity of the selected compounds derived from the inhibition of DNA gyrase, which is consistent with previously reported NBTIs [1] where DNA gyrase inhibition in S. aureus is superior to topoisomerase IV. Among those compounds, compound 9 demonstrated the best enzymatic inhibition for both DNA gyrase (IC50=0.10

M) and topoisomerase IV (IC50=14 M), but efforts are likely still needed to improve topoisomerase IV inhibitory activity to reduce the rate of resistance emergence.

Table 2.4 Target inhibition of selected compounds Compound S. aureus S. aureus Compound S. aureus S. aureus DNA gyrase topoisomerase DNA gyrase topoisomerase IC50 (M) IV IC50 (M) IC50 (M) IV IC50 (M) (n=2)a (n=2)b (n=2)a (n=2)b

9 0.10 14 20 0.28 >100

10 0.36 50 25 1.2 >100

11 0.33 25-50 27 0.22 >100

12 0.59 >100 28 0.3 ~100

13 0.78 >100 29 1.5 >100

14 0.74 >100 33 0.63 >100

15 0.52 >100 36 0.36 >100

16 0.17 74 45 0.95 >100

17 NT >200c 55 0.81 13

19 NT >200c ciprofloxacin 4.80 (n=4) 14.3 (n=4)

aSupercoiling inhibition assay, Inspiralis, Ltd. (Norwich, UK). bDecatenation inhibition assay, Inspiralis, Ltd. (Norwich, UK).

2.3.2.3 Preliminary in vitro safety evaluation

As the key hypothesis of this work, the 5-amino-1,3-dioxane linker in our novel NBTIs was postulated to attenuate hERG toxicity due to its reduced basicity and enhanced polarity resulting from the two electronegative oxygen atoms [26, 27]. Two series of matched pair compounds were employed to evaluate this idea: compounds 91-94 used a cyclohexane ring as the linker moiety while compounds 100-103 contained the piperidine linker. Data from these compounds were compared with results obtained from the analogous dioxane- linked NBTIs (9, 10, 13, and 20).

IC50 values for hERG inhibition were determined by Charles River (Cleveland, OH).

As shown in Table 2.5, all matched dioxane/cyclohexane pairs (9/91, 10/92, 13/93, 20/94) manifested the superiority of the dioxane linker, with 2- to 20-fold improvements. The matched pair compounds 9/91 displayed the greatest disparity. The hERG inhibition of the dioxane-linked analog 9 (IC50=5.11M) was 20-fold less than that of the corresponding cyclohexane-linked analog 91 (IC50=0.26M). Three of four dioxane-piperidine pairs (9/100, 10/101, 13/102, 20/103) also demonstrated improvements (5- to 6-fold) for the dioxane linker. The only exception was the pyridooxazinone 13 (IC50=2.5M), which was slightly inferior to the piperidine-linked analog 102 (IC50=3.8M). In accordance with previous reports [19, 20], pyridothiazinone (12) or pyridooxazinone (13) enzyme-binding moiety generally showed potent hERG inhibition. Moreover, the more polar pyridodioxine moiety proved consistently superior to the more lipophilic benzodioxine moiety (ca. ~2- fold improvement in each series). In summary, the hERG inhibition results gave strong support for the design hypothesis that the 5-amino-1,3-dioxane linker moiety would reduce hERG inhibition by NBTIs. Nevertheless, additional improvements are definitely necessary to obtain compounds with desirable further reductions in hERG inhibition (IC50 > 100M). Our further efforts to address this objective will be discussed in Chapter 3.

Table 2.5 In vitro safety evaluation data of selected NBTIs

b,c Compound Linker hERG IC50 K562 K/VP.5 hTopoII a (M) IC50 IC50 (M) (M)

9 dioxane 5.1 7.4 8.8 84.18

10 dioxane 9.5 40 48 95.12

11 dioxane 12 32 40. 93.81

12 dioxane 1.3 31 34 91.14

13 dioxane 2.5 24 44 99.06

15 dioxane 5.0 14 14 NTd

16 dioxane 8.6 25 26 NT

20 dioxane 4.8 20 15 93.22

25 dioxane 2.0 12 16 97.84

27 dioxane 4.4 17 25 NT

28 dioxane 3.4 8.9 12 NT

30 dioxane 3.2 13 14 NT

33 dioxane 4.7 7.6 6.3 NT

55 dioxane 12 28 20 103.1

91 cyclohexane 0.26 4.2 4.5 NT

92 cyclohexane 0.94 7.5 5.7 NT

93 cyclohexane 0.41 12 11 NT

94 cyclohexane 2.3 NT NT NT

100 piperidine 0.81 10. 12 NT

101 piperidine 1.9 28 22 NT

102 piperidine 3.8 19 16 NT

103 piperidine 1.0 NT NT NT

Cisapridee NA 0.012-0.021 NT NT NT

etoposide NA NT 0.3 5.1 33.6 (n=4) ciprofloxacin NA NT NT NT 85.5 (n=4) aDetermined using IonWorks Barracuda at Charles River (Cleveland, OH). bInspiralis, Ltd. (Norwich, UK). cPercent enzyme activity remaining at 100 M. dNT = not tested. eCisapride serves as the positive control for the hERG study.

Bacterial and eukaryotic type II topoisomerases are orthologous, but DNA gyrase is essential to bacterial cells and absent in eukaryotes [37]. The eukaryotic Topo II is a large homodimer, while bacterial topoisomerase II is a tetramer assembled from two subunits, such as GyrA2GyrB2 for DNA gyrase and ParC2ParE2 for topoisomerase IV. The major structural difference between them is that the C-terminal domain of bacterial GyrA or ParE adopts a unique β-pinwheel fold with DNA-binding activity, whereas the C-terminal tails 76 of eukaryotic enzymes are likely unstructured [37]. Moreover, the most common bacterial mutations in quinolone-resistant strains are the conserved gyrase serine and glutamic acid residues (S85 and E89 in B. anthracis gyrase), which differ from humans [38, 39]. Due to these structural and sequence distinctions, selectivity over the human targets is expected for NBTIs but still needs to be evaluated.

In order to test the selectivity over human targets, these new potent compounds above were assayed in antiproliferative experiments using human leukemia K562 cells and an isogenic (etoposide-resistant) subline, K/VP.5 [40], which contains 1/5th the level of hTopoII compared to the parental K562 cells. These antiproliferative assays were carried out in Dr. Yalowich’s lab at The Ohio State University, with results shown in table 2.5. Etoposide worked as positive control and demonstrated a 17-fold loss of inhibition in

K/VP.5 as compared to K652 cells (Table 2.5). In contrast, the growth inhibitory IC50 values for the selected compounds against K/VP.5 versus K652 cells were nearly identical, strongly suggesting that the new NBTIs do not target human TopoII. To substantiate this argument, numerous compounds were also assayed for inhibition of enzymatic activity using isolated human topoisomerase II [note, no inhibition = 100% remaining activity]. Compared with the positive control etoposide (33.6% remaining activity), all of the new compounds preserved high human TopoII activity (>90%), even at the high concentration of 100M. Thus, both the antiproliferative assays and the human topoisomerase II biochemical assays revealed minimal inhibition of the human enzyme by these new NBTIs.

2.4 Conclusions

In this chapter, we employed a 5-amino-1,3-dioxane moiety as the linker domain in the design of new NBTIs. A series of 6-methoxyquinoline analogs (compound 9-57) were synthesized via uncomplicated and economical steps. Required intermediates that were not commercially available materials were prepared from inexpensive starting materials. Meanwhile, several structure-matched compounds with cyclohexane (91-94) and piperidine (100-103) linkers were synthesized for comparison.

The SAR study of the 6-methoxyquinoline series showed that compounds with a lipophilic para-substituented monocyclic enzyme-binding moiety have moderate anti- staphylococcal activity, while compounds with bicyclic enzyme-binding moieties generally possess more potent whole cell anti-staphylococcal activity. These dioxane- linked NBTIs also retained good activity against a ciprofloxacin-resistant strain of MRSA. 6-methoxyquinoline derivatives with a vinyl group in the linker did not possess improved potency. Studies of cell-free enzyme inhibition revealed that the whole cell antibacterial activity could be attributed to the inhibition of DNA gyrase rather than topoisomerase IV, which is in accordance with results from previously reported NBTIs.

To our delight and consistent with the design hypothesis, hERG inhibition experiments showed that our dioxane-linked NBTIs demonstrate reduced hERG inhibition as compared with structure-matched NBTIs with cyclohexane or piperidine linkers. Meanwhile, these new NBTIs showed minimal inhibition of human topoisomerase II and low toxicity to human cells. In this initial work, compound 9 presented the best results in terms of whole cell activity (0.25-1 g/mL for MSSA, 0.5 g/mL for MRSA), and bacterial topoisomerase inhibition (DNA gyrase IC50=0.10 M and topoisomerase IV IC50=14 M). The more polar compound 55 displayed the best hERG profile (IC50=12 M) and minimal off-target effects on the human homologous enzyme. More endeavors to enhance antibacterial activity, to improve topoisomerase IV inhibition, and to reduce hERG inhibition further should be made. Our progress toward these goals is presented in Chapter 3.

2.5 Experimental Section

2.5.1 Chemistry part

General Chemistry Information. Moisture and/or air-sensitive reactions were conducted in oven-dried glassware under an atmosphere of nitrogen or argon unless otherwise noted. Dichloromethane (DCM), toluene, tetrahydrofuran (THF), and N,N-dimethylformamide (DMF) were dried before use by passage through activated alumina under nitrogen. Flash chromatography was performed using a Teledyne-ISCO Combiflash-Rf+. 1H NMR spectra

78 were recorded at either 300 MHz or 400 MHz using residual protiated solvent as the internal reference: CDCl3 (7.26 ppm), CD3OD (3.31), DMSO-d6 (2.50 ppm). Assayed compounds had a purity of >90% as determined by 1H NMR analysis. 13C NMR spectra were recorded at 75 MHz or 100 MHz using the solvent signal as the internal reference:

CDCl3 (77.16 ppm), CD3OD (49.00), DMSO-d6 (39.52 ppm). High resolution mass spectrometry was performed using electrospray ionization. The equatorial protons at C4 and C6 of the trans-dioxane ring typically appear as an apparent doublet of doublets at ca. 4.1-4.2 ppm in the analogs described below.[41] Careful inspection in some cases reveals additional partially resolved splitting, arising from W-coupling of these magnetically non- equivalent protons as well as virtual coupling. The additional splitting can be effectively replicated using computer simulation of non-first order effects [42], using a W-coupling constant of 1.8 Hz. For the sake of clarity and consistency, this peak is labeled a doublet of doublets in the characterization data provided below.

4-bromo-6-methoxyquinoline (2). A three-necked round bottom flask vented to a 1M NaOH (aq) gas trap was charged with 4-hydroxy-6-methoxyquinoline (12.07 g, 68.90 mmol, 1.0 equiv) and anhydrous N,N-dimethylformamide (75 mL) and stirred magnetically. To the heterogeneous mixture was added PBr3 (8.0 mL, 85 mmol, 1.2 equiv) drop/portionwise over several minutes by syringe. During the addition, the internal temperature rose to 75 °C, and gas was evolved. A copious precipitate formed toward the end of the addition. The vent to the gas trap was 15 min after the completion of addition, and the reaction was stirred vigorously for an additional 1h 45 min, then quenched by pouring onto 150 g ice in 150 mL water. After brief stirring, Na2CO3 (20 g) was added in small portions, and the mixture was stirred for 15 min, whereupon the pH was approximately 7. The taupe-colored product was isolated by vacuum filtration on a

Buchner funnel, washing extensively with water. After drying for several days in a vacuum desiccator, the title compound was obtained in ca. 95% purity as a light tan, powdery solid (15.58 g, 65.44 mmol, 95%).

2-(1,3-dihydroxypropan-2-yl)isoindoline-1,3-dione (5). Serinol 3 (35.10 g, 38.52 mmol, 1.0 equiv) and phthalic anhydride 4 (58.18 g, 39.28 mmol, 1.0 equiv) were suspended in anhydrous toluene (500 mL), stirred, and heated at 105 °C (internal temperature) for 24 h. The heat was then removed, and a precipitate rapidly formed when the temperature reached 80 °C. After the temperature had reached 62 °C, Methyl tert-butyl ether (MTBE) was added, and the mixture was mechanically stirred for 1 h. Vacuum filtration on a Buchner funnel afforded an off-white powder with light yellow solid clumps that were ground using a mortar and pestle. The combined solids were then suspended in MTBE and stirred for 2 h, then re-isolated by vacuum filtration and dried to afford the title compound in ca. 95% purity as an off-white powder (77.84 g, 35.19 mmol, 91%). 1H NMR (300 MHz, DMSO- d6) δ: 7.88-7.80 (m, 4H); 4.85 (br t, J = 6.0, 2H); 4.24 (dddd, J = 5.7, 5.7, 8.7, 8.7, 1H); 3.80 (ddd, J = 5.4, 8.8, 11.2, 2H); 3.71-3.61 (ddd, J = 6.0, 6.0, 11.2, 2H).

2-(2-vinyl-trans-1,3-dioxan-5-yl)isoindoline-1,3-dione (6). A mixture of acrolein (4.16 mL, 62.3 mmol, 3.1 equiv), diol 5 (4.426 g, 20.01 mmol, 1.0 equiv), p-toluenesulfonic acid (0.12 g, 0.63 mmol, 0.03 equiv), anhydrous cupric sulfate (1.6 g, 10. mmol, 0.5 equiv) and anhydrous DCM (60 mL) was stirred at room temperature under N2 atmosphere for 36 h.

The mixture was then washed with brine, and the aqueous phase was extracted with DCM. The combined organic layers were concentrated, and the crude product was purified by chromatography on silica gel with DCM/methanol (50:1) to give the title compound as a 1 white solid (3.948 g, 15.23 mmol, 76%). H NMR (300 MHz, CDCl3) δ: 7.88-7.81 (m, 2H); 7.77-7.70 (m, 2H) 5.90 (ddd, J = 4.5, 10.7, 17.4, 1H); 5.53 (d apparent t, J = 1.2, 17.4, 1H); 5.36 (d apparent t, J = 1.1, 10.7, 1H); 5.10 (br d, J = 4.5, 1H); 4.73-4.61 (m, 1H); 4.55-4.46 (m, 2H); 4.09 (dd, J = 4.7, 10.6, 2H).

2-(2-(2-(6-methoxyquinolin-4-yl)ethyl)-trans-1,3-dioxan-5-yl)isoindoline-1,3-dione (7). To a solution of alkene 6 (2.312 g, 8.918 mmol, 1.1 equiv) in THF (18 mL) was added 9-borabicyclo[3.3.1]nonane (0.5 M in THF, 16.6 mL, 8.3 mmol, 1.0 equiv) dropwise at room temperature under N2 atmosphere. The mixture was stirred for 3 hours and used directly in the next step. To a mixture of compound 2 (1.666 g, 6.998 mmol, 1.0 equiv), cesium carbonate (5.700 g, 17.49 mmol, 2.5 equiv), bis(triphenylphosphine)palladium(II) dichloride (0.2315 g, 0.3298 mmol, 0.05 equiv) and THF (40 mL), the solution from above was added slowly at room temperature under N2 atmosphere. The reaction mixture was stirred overnight. Purification by chromatography on silica gel with DCM/methanol (50:1) afforded the title compound as a white solid (1.660 g, 3.8mmol, 54%). 1H NMR (300 MHz,

CDCl3) δ: 8.69 (d, J = 4.5, 1H); 8.05 (d, J = 9.0, 1H); 7.89-7.81 (m, 2H); 7.78-7.71 (m, 2H); 7.38 (dd, J = 2.7, 9.1, 1H); 7.34 (d, J = 2.6, 1H); 7.24 (d, partially obscured by solvent, 1H); 4.78 (t, J = 4.7, 1H); 4.74-4.61 (m, 1H); 4.46 (t, J = 11.1, 2H); 4.08 (dd, J = 4.8, 10.7, 2H), 3.97 (s, 3H); 3.24-3.16 (m, 2H), 2.19-2.10 (m, 2H).

2-(2-(6-methoxyquinolin-4-yl)ethyl)-trans-1,3-dioxan-5-amine (8). A mixture of compound 7 (1.312 g, 3.135 mmol, 1.0 equiv), ethanolamine (2.73 mL, 45.2 mmol, 14 equiv) and ethyl acetate (40 mL) was stirred and heated at 70 °C overnight. The solvent was removed, and the mixture was dissolved in DCM and washed with brine. The organic layer was combined and concentrated, and the crude product was purified by chromatography on silica gel with DCM/methanol (15:1) to give the title compound as an 1 oil (0.679 g oil, 2.35 mmol, 75%). H NMR (300 MHz, CDCl3) δ: 8.65 (d, J = 4.5, 1H); 7.99 (d, J = 9.1, 1H); 7.35 (dd, J = 2.8, 9.1, 1H); 7.30 (d, J = 2.7, 1H); 7.19 (d, J = 4.5, 1H); 4.44 (t, J = 4.9, 1H); 4.15 (dd, J = 4.5, 10.8, 2H); 3.94 (s, 3H); 3.27-3.03 (m, 5H); 2.11- 13 2.01 (m, 2H), 1.24 (br s, 2H). C NMR (75 MHz, CDCl3) δ: 157.8, 147.8, 146.2, 144.4, 131.7, 128.5, 121.6, 121.1, 101.9, 100.7, 73.5, 55.6, 44.3, 34.2, 26.5. HRMS (ESI) m/z + calc’d for C16H21N2O3 [M+H] : 289.1552; found: 289.1551.

Reductive amination general procedure: To a solution of amine (0.2 mmol) in methanol (2 mL) was added the requisite aldehyde (0.2 mmol) and zinc chloride (2 mg, 0.015 mmol, 0.07 equiv). The mixture was stirred at room temperature for 30 min, followed by addition of sodium cyanoborohydride (40 mg, 0.6 mmol, 3.0 equiv). The reaction mixture was stirred at room temperature overnight and then purified by chromatography on silica gel with DCM/methanol (50:1). Occasionally, the product obtained from flash chromatography was contaminated by a BH3 adduct, seen in the 1H NMR spectrum as four

82 broad peaks from 0.00-0.88 ppm. In these instances, the column-purified material was dissolved in methanol (2 mL) and stirred overnight at ambient temperature with Amberlite IRA743 free base (ca. 100 mg). The pure title compound was then obtained by removal of the resin by filtration and removal of the solvent under reduced pressure.

The following dioxane-linked compounds were prepared from amine 8 using the general reductive amination procedure:

N-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-2-(2-(6-methoxyquinolin-4- yl)ethyl)-trans-1,3-dioxan-5-amine (9). The title compound was prepared in 60 % yield 1 following the general method. H NMR (300 MHz, CDCl3) δ: 8.65 (d, J = 4.4, 1H); 8.00 (d, J = 9.1, 1H); 7.35 (dd, J = 2.7, 9.1, 1H); 7.30 (d, J = 2.7, 1H); 7.18 (d, J = 4.4, 1H); 6.81 (d, J = 8.2, 1H); 6.81 (d, J = 1.8, 1H); 6.75 (dd, J = 1.9, 8.2, 1H); 4.46 (t, J = 4.8, 1H); 4.23 (s, 4H); 4.19 (dd, J = 4.7, 11.2, 2H); 3.93 (s, 3H); 3.69 (s, 2H); 3.28 (t, J = 10.8, 2H); 3.17-3.07 (m, 2H); 3.05-2.93 (m, 1H); 2.11-2.00 (m, 2H); 1.03 (br s, 1H). 13C NMR (75

MHz, CDCl3) δ: 157.8, 147.8, 146.2, 144.5, 143.6, 142.9, 133.6, 131.8, 128.5, 121.5, 121.1, 121.0, 117.4, 116.9, 101.9, 101.0, 71.7, 64.49, 64.46, 55.7, 50.9, 49.8, 34.3, 26.4. HRMS + (ESI) m/z calc’d for C25H29N2O5 [M+H] : 437.2076; found: 437.2039.

N-((2,3-dihydro-[1,4]dioxino[2,3-c]pyridin-7-yl)methyl)-2-(2-(6-methoxyquinolin-4- yl)ethyl)-trans-1,3-dioxan-5-amine (10). The title compound was prepared in 70 % yield

1 following the general method. H NMR (300 MHz, CDCl3) δ: 8.63 (d, J = 4.4, 1H); 8.08 (s, 1H); 7.98 (d, J = 9.1, 1H); 7.33 (dd, J = 2.7, 9.1, 1H); 7.28 (d, J = 2.6, 1H); 7.17 (d, J = 4.3, 1H); 6.77 (s, 1H); 4.47 (t, J = 4.8, 1H); 4.33-4.16 (m, 6H); 3.92 (s, 3H); 3.77 (s, 2H); 3.33 (t, J = 10.6, 2H); 3.15-3.06 (m, 2H); 3.04-2.90 (m, 1H); 2.11-1.98 (m, 2H); 1.95 (br s, 13 1H). C NMR (75 MHz, CDCl3) δ: 157.8, 152.9, 150.4, 147.8, 146.3, 144.4, 140.4, 139.0, 131.7, 128.5, 121.5, 121.0, 110.7, 101.9, 101.0, 71.6, 65.1, 64.1, 55.6, 52.0, 50.1, 34.3, + 26.4. HRMS (ESI) m/z calc’d for C24H28N3O5 [M+H] : 438.2029; found: 438.1992.

N-((3,4-dihydro-2H-pyrano[2,3-c]pyridin-6-yl)methyl)-2-(2-(6-methoxyquinolin-4- yl)ethyl)-trans-1,3-dioxan-5-amine (11). The title compound was prepared in 54 % yield 1 following the general method. H NMR (300 MHz, CDCl3) δ: 8.63 (d, J = 4.5, 1H); 8.06 (s, 1H); 7.99 (d, J = 9.2, 1H); 7.36 (dd, J = 2.7, 9.2, 1H); 7.29 (d, J = 2.6, 1H); 7.21 (d, J = 4.5, 1H); 6.95 (s, 1H); 4.50 (t, J = 4.8, 1H); 4.27-4.17 (m, 4H); 3.93 (s, 3H); 3.83 (s, 2H); 3.41 (t, J = 10.8, 2H); 3.17-3.08 (m, 2H); 3.08-2.97 (m, 1H); 2.75 (t, J = 6.4, 2H); 2.10- 13 1.95 (m, 4H). C NMR (75 MHz, CDCl3) δ: 158.0, 151.4, 148.9, 147.2 (two overlapping signals), 143.6, 138.7, 131.7, 131.0, 128.6, 123.1, 122.0, 121.1, 101.9, 101.0, 71.1, 66.7, + 55.7, 51.5, 50.1, 34.1, 26.5, 24.3, 21.6. HRMS (ESI) m/z calc’d for C25H30N3O4 [M+H] : 436.2236; found: 436.2210.

6-(((2-(2-(6-methoxyquinolin-4-yl)ethyl)-trans-1,3-dioxan-5-yl)amino)methyl)-2H- pyrido[3,2-b][1,4]thiazin-3(4H)-one (12). The title compound was prepared in 46 % yield 1 following the general method. H NMR (300 MHz, CDCl3) δ: 8.66 (d, J = 4.5, 1H); 8.57 (br s, 1H); 8.02 (d, J = 9.2, 1H); 7.58 (d, J = 7.8, 1H); 7.35 (dd, J = 2.8, 9.2, 1H); 7.29 (d, J = 2.7, 1H); 7.19 (d, J = 4.5, 1H); 6.95 (d, J = 7.8, 1H); 4.47 (t, J = 4.9, 1H); 4.22 (dd, J = 4.7, 11.2, 2H); 3.94 (s, 3H); 3.84 (s, 2H); 3.47 (s, 2H); 3.34 (t, J = 10.9, 2H); 3.17-3.08 (m, 13 2H); 3.04-2.92 (m, 1H); 2.11-2.01 (m, 2H). C NMR (75 MHz, CDCl3) δ: 165.7, 157.9, 156.7, 148.4, 147.8, 146.3, 144.5, 136.4, 131.7, 128.6, 121.5, 121.1, 117.9, 114.0, 102.0,

101.1, 71.6, 55.7, 51.7, 50.2, 34.3, 29.8, 26.5. HRMS (ESI) m/z calc’d for C24H27N4O4S [M+H]+: 467.1753; found: 467.1727.

6-(((2-(2-(6-methoxyquinolin-4-yl)ethyl)-trans-1,3-dioxan-5-yl)amino)methyl)-2H- pyrido[3,2-b][1,4]oxazin-3(4H)-one (13). The title compound was prepared in 35 % yield 1 following the general method. H NMR (300 MHz, CDCl3) δ: 8.66 (d, J = 4.4, 1H); 8.64 (br s, 1H); 8.02 (d, J = 9.2, 1H); 7.36 (dd, J = 2.7, 9.1, 1H); 7.34 (d, J = 2.7, 1H); 7.22 (d, J = 8.1, 1H); 7.19 (d, J = 4.6, 1H); 6.92 (d, J = 8.1, 1H); 4.65 (s, 2H); 4.47 (t, J = 4.9, 1H); 4.22 (dd, J = 4.7, 11.2, 2H); 3.94 (s, 3H); 3.82 (s, 2H); 3.34 (t, J = 10.9, 2H); 3.17-3.08 (m, 13 2H); 3.06-2.91 (m, 1H); 2.12-2.01 (m, 2H). C NMR (75 MHz, CDCl3) δ: 165.4, 157.9, 151.6, 147.8, 146.3, 144.5, 140.3, 138.5, 131.7, 128.6, 124.4, 121.5, 121.1, 118.2, 102.0,

101.1, 71.6, 67.4, 55.7, 51.5, 50.2, 34.3, 26.5. HRMS (ESI) m/z calc’d for C24H27N4O5 [M+H]+: 451.1981; found: 451.1970.

6-(((2-(2-(6-methoxyquinolin-4-yl)ethyl)-trans-1,3-dioxan-5-yl)amino)methyl)-2H- benzo[b][1,4]oxazin-3(4H)-one (14). The title compound was prepared in 71 % yield 1 following the general method. H NMR (300 MHz, CDCl3) δ: 9.16 (br s, 1H); 8.66 (d, J = 4.5, 1H); 8.01 (d, J = 9.2, 1H); 7.35 (dd, J = 2.7, 9.2, 1H); 7.29 (d, J = 2.6, 1H); 7.19 (d, J = 4.5, 1H); 6.95-6.86 (m, 2H); 6.80 (br s, 1H), 4.61 (s, 2H); 4.46 (t, J = 4.8, 1H); 4.19 (dd, J = 4.6, 11.0, 2H); 3.93 (s, 3H); 3.72 (s, 2H); 3.28 (t, J = 10.8, 2H); 3.17-3.07 (m, 2H); 13 3.04-2.91 (m, 1H); 2.11-2.00 (m, 2H). C NMR (75 MHz, CDCl3) δ: 166.3, 157.8, 147.8, 146.2, 144.5, 143.0, 135.1, 131.7, 128.6, 126.3, 123.8, 121.5, 121.1, 116.9, 115.7, 102.0,

101.1, 71.7, 67.4, 55.7, 50.7, 49.9, 34.3, 26.5. HRMS (ESI) m/z calc’d for C25H28N3O5 [M+H]+: 450.2029; found: 450.2004.

N-((2,2-difluorobenzo[d][1,3]dioxol-5-yl)methyl)-2-(2-(6-methoxyquinolin-4- yl)ethyl)-trans-1,3-dioxan-5-amine (15). The title compound was prepared in 53 % yield 1 following the general method and obtained as white solid. H NMR (300 MHz, CDCl3) δ: 8.66 (d, J=4.4 Hz, 1H); 8.01 (d, J=9.1 Hz, 1H); 7.36 (dd, J=9.1, 2.8 Hz, 1H); 7.29 (d, J=2.7 Hz, 1H); 7.19 (d, J=4.5 Hz, 1H); 7.08 (s, 1H); 6.99 (s, 2H); 4.46 (t, J=4.9 Hz, 1H); 4.20 (dd, J=11.1, 4.8 Hz, 2H); 3.94 (s, 3H); 3.80 (s, 2H); 3.29 (t, J=10.9 Hz, 2H); 3.12 (t, J=7.8 13 Hz, 2H); 3.02-2.92 (m, 1H); 2.09-2.02 (m, 2H). C NMR (75 MHz, CDCl3) δ: 162.5, 157.9, 147.8, 146.3, 144.4, 144.2, 143.1, 136.6, 131.7, 128.6, 123.0, 121.6, 121.1, 109.35, 109.30,

102.0, 101.1, 71.6, 55.7, 51.0, 49.9, 34.3, 26.5. HRMS (ESI) m/z calc’d for C24H25F2N2O5 [M+H]+: 459.1732. found: 459.1742.

N-(benzo[d][1,3]dioxol-5-ylmethyl)-2-(2-(6-methoxyquinolin-4-yl)ethyl)-trans-1,3- dioxan-5-amine (16). The title compound was prepared in 58 % yield following the 1 general method and obtained as light yellow solid. H NMR (300 MHz, CDCl3) δ: 8.65 (d, J=4.4 Hz, 1H); 8.00 (d, J=9.1 Hz, 1H); 7.35 (dd, J=9.1, 2.8 Hz, 1H); 7.29 (d, J=2.7 Hz, 1H); 7.18 (d, J=4.5 Hz, 1H); 6.81 (s, 1H); 6.74 (s, 2H); 5.93 (s, 2H); 4.46 (t, J=4.9 Hz, 1H); 4.19 (dd, J=10.8, 4.5 Hz, 2H); 3.93 (s, 3H); 3.71 (s, 2H); 3.28 (t, J=10.9 Hz, 2H); 3.11 (t, J=7.8 Hz, 2H); 3.03-2.93 (m, 1H); 2.11-2.00 (m, 2H). 13C NMR (75 MHz,

CDCl3) δ: 157.8, 148.0, 147.8, 146.9, 146.2, 144.5, 134.2, 131.8, 128.6, 121.5, 121.2, 121.1, 108.6, 108.3, 101.9, 101.1, 101.0, 71.7, 55.6, 51.3, 49.8, 34.3, 26.4. HRMS (ESI) + m/z calc’d for C24H27N2O5 [M+H] : 423.1920. found: 423.1936.

2-(2-(6-methoxyquinolin-4-yl)ethyl)-N-(naphthalen-2-ylmethyl)-trans-1,3-dioxan-5- amine (17). The title compound was prepared in 61 % yield following the general 1 method and obtained as white solid. H NMR (300 MHz, CDCl3) δ: 8.65 (d, J=4.4 Hz, 1H); 8.01 (d, J=9.1 Hz, 1H); 7.83-7.79 (m, 3H); 7.74 (s, 1H); 7.52-7.41 (m, 3H); 7.35 (dd, J=9.1, 2.8 Hz, 1H); 7.29 (d, J=2.7 Hz, 1H); 7.18 (d, J=4.5 Hz, 1H); 4.47 (t, J=4.9 Hz, 1H); 4.24 (dd, J=11.1, 4.7 Hz, 2H); 3.98 (s, 2H); 3.91 (s, 3H); 3.33 (t, J=10.9 Hz, 13 2H); 3.17-2.99 (m, 3H); 2.11-1.99 (m, 2H). C NMR (75 MHz, CDCl3) δ: 157.8, 147.8, 146.3, 144.5, 137.7, 133.5, 132.9, 131.7, 128.5, 128.4, 127.8, 126.5, 126.3, 126.3, 125.9,

121.5, 121.1, 101.9, 101.0, 71.7, 55.6, 51.7, 50.0, 34.3, 26.4. HRMS (ESI) m/z calc’d for + C27H29N2O3 [M+H] : 429.2178. found: 429.2184.

2-(2-(6-methoxyquinolin-4-yl)ethyl)-N-(naphthalen-1-ylmethyl)-trans-1,3-dioxan-5- amine (18). The title compound was prepared in 60 % yield following the general method 1 and obtained as white solid. H NMR (300 MHz, CDCl3) δ: 8.66 (d, J=4.4 Hz, 1H); 8.08 (dd, J=7.9, 1.1 Hz, 1H); 8.01 (d, J=9.1 Hz, 1H); 7.87 (dd, J=7.3, 2.1 Hz, 1H); 7.79 (dd, J=7.5, 1.7 Hz, 1H); 7.58-7.39 (m, 4H); 7.36 (dd, J=9.1, 2.8 Hz, 1H); 7.30 (d, J=2.7 Hz, 1H); 7.19 (d, J=4.5 Hz, 1H); 4.48 (t, J=4.9 Hz, 1H); 4.30-4.23 (m, 4H); 3.93 (s, 3H); 3.34 13 (t, J=10.8 Hz, 2H); 3.20-3.07 (m, 3H); 2.13-2.02 (m, 2H). C NMR (75 MHz, CDCl3) δ: 157.8, 147.8, 146.3, 144.5, 135.6, 134.1, 131.7, 129.0, 128.6, 128.3, 126.34, 126.32, 125.9, 125.5, 123.6, 121.6, 121.1, 101.9, 101.1, 71.7, 55.7, 50.6, 49.5, 34.3, 26.5. HRMS (ESI) + m/z calc’d for C27H29N2O3 [M+H] : 429.2178. found: 429.2181.

N-((1,2-dihydroacenaphthylen-5-yl)methyl)-2-(2-(6-methoxyquinolin-4-yl)ethyl)- trans-1,3-dioxan-5-amine (19). The title compound was prepared in 64 % yield following 1 the general method and obtained as light yellow solid. H NMR (300 MHz, CDCl3) δ: 8.66 (d, J=4.4 Hz, 1H); 8.01 (d, J=9.1 Hz, 1H); 7.73 (d, J=8.3 Hz, 1H); 7.48 (dd, J=8.3, 6.9 Hz, 1H); 7.41-7.33 (m, 2H); 7.33-7.28 (m, 2H); 7.24 (d, J=7.1 Hz, 1H); 7.20 (d, J=4.5 Hz, 1H); 4.48 (t, J=4.9 Hz, 1H); 4.24 (dd, J=11.1, 4.7 Hz, 2H); 4.20 (s, 2H); 3.93 (s, 3H); 3.46-3.27 13 (m, 6H); 3.19-3.05 (m, 3H); 2.14-2.01 (m, 2H). C NMR (75 MHz, CDCl3) δ: 157.8, 147.8, 88

146.8, 146.3, 146.0, 144.5, 139.8, 131.7, 131.6, 130.2, 128.6, 128.1, 127.9, 121.5, 121.1, 119.5, 119.0, 119.0, 101.9, 101.1, 71.8, 55.7, 50.4, 48.7, 34.3, 30.7, 30.1, 26.5. HRMS + (ESI) m/z calc’d for C29H31N2O3 [M+H] : 455.2335. found: 455.2339.

N-(3,4-dichlorobenzyl)-2-(2-(6-methoxyquinolin-4-yl)ethyl)-trans-1,3-dioxan-5- amine (20). The title compound was prepared in 58 % yield following the general method 1 and obtained as white solid. H NMR (300 MHz, CDCl3) δ: 8.66 (br s, 1H); 8.02 (d, J = 9.1 Hz, 1H); 7.41 (d, J = 1.9 Hz, 1H); 7.37 (d, J = 8.1 Hz, 1H); 7.35 (dd, J = 9.1, 2.8 Hz, 1H); 7.28 (d, J = 2.6 Hz, 1H); 7.18 (d, J = 4.2 Hz, 1H); 7.13 (dd, J = 8.2, 2.0 Hz, 1H); 4.47 (t, J = 4.8 Hz,1H); 4.19 (dd, J = 11.2, 4.6 Hz, 2H); 3.94 (s, 3H); 3.78 (s, 2H); 3.28 (t, J = 10.9 Hz, 2H); 3.13 (t, J = 8.0 Hz, 2H); 3.00-2.93 (m, 1H); 2.10-2.03 (m, 2H). 13C NMR

(75 MHz, CDCl3) δ: 157.8, 147.8, 146.2, 144.5, 140.7, 132.7, 131.7, 131.2, 130.5, 129.9, 128.5, 127.3, 121.5, 121.1, 101.9, 101.0, 71.6, 55.6, 50.3, 50.0, 34.2, 26.4. HRMS (ESI) + m/z calc’d for C23H25Cl2N2O3 [M+H] : 447.1242; found: 447.1235.

N-((5-chloropyridin-2-yl)methyl)-2-(2-(6-methoxyquinolin-4-yl)ethyl)-trans-1,3- dioxan-5-amine (24). The title compound was prepared in 55 % yield following the 1 general method and obtained as white solid. H NMR (300 MHz, CDCl3) δ: 8.65 (d, J = 4.5 Hz, 1H); 8.51 (d, J = 2.2 Hz, 1H); 8.03 (d, J = 9.2 Hz, 1H); 7.63 (dd, J = 8.3, 2.5 Hz, 1H); 7.38 (dd, J = 9.2, 2.7 Hz, 1H); 7.31 (d, J = 2.7 Hz, 1H); 7.25 (d, J = 8.0, 1H); 7.23 (d, J = 4.6 Hz, 1H); 4.49 (t, J = 4.8 Hz, 1H); 4.23 (dd, J = 11.2, 4.7 Hz, 2H); 3.95 (s, 3H); 3.92 (s, 2H); 3.35 (t, J = 10.9 Hz, 2H); 3.14 (t, J = 7.8 Hz, 2H); 3.04-2.94 (m, 1H); 2.10-2.03 13 (m, 2H). C NMR (75 MHz, CDCl3) δ: 157.9, 157.4, 148.2, 147.1, 147.0, 143.6, 136.3, 89

131.0, 130.6, 128.5, 122.9, 121.9, 121.0, 101.9, 100.9, 71.5, 55.6, 51.8, 50.2, 34.1, 26.4. + HRMS (ESI) m/z calc’d for C22H25ClN3O3 [M+H] : 414.1584; Found: 414.1577.

2-(2-(6-methoxyquinolin-4-yl)ethyl)-N-(4-(trifluoromethyl)benzyl)-trans-1,3-dioxan- 5-amine (28). The title compound was prepared in 54 % yield following the general 1 method and obtained as white solid. H NMR (300 MHz, CDCl3) δ: 8.66 (d, J=4.4 Hz, 1H); 8.01 (d, J=9.1 Hz, 1H); 7.59 (br d, J=8.1 Hz, 2H); 7.43 (br d, J=8.0 Hz, 2H); 7.36 (dd, J=9.1, 2.8 Hz, 1H); 7.30 (d, J=2.7 Hz, 1H); 7.19 (d, J=4.5 Hz, 1H); 4.47 (t, J=4.9 Hz, 1H); 4.21 (dd, J=11.1, 4.7 Hz, 2H); 3.94 (s, 3H); 3.88 (s, 2H); 3.30 (t, J=10.8 Hz, 2H); 3.12 (t, 13 J=7.8 Hz, 2H); 3.04-2.94 (m, 1H); 2.10-2.03 (m, 2H). C NMR (75 MHz, CDCl3) δ: 157.9, 147.9, 146.2, 144.5, 144.4, 131.8, 130.0, 129.5, 128.6, 128.3, 125.5 (q, J=3.6Hz), 122.5, 121.5, 121.1, 102.0, 101.1, 71.7, 55.7, 51.0, 50.1, 34.3, 26.5. HRMS (ESI) m/z calc’d for + C24H26F3N2O3 [M+H] : 447.1895. found: 447.1867.

N-(4-methoxybenzyl)-2-(2-(6-methoxyquinolin-4-yl)ethyl)-trans-1,3-dioxan-5-amine (29). The title compound was prepared in 70 % yield following the general method and 1 obtained as white solid. H NMR (300 MHz, CDCl3) δ: 8.65(d, J=4.5 Hz, 1H); 8.00 (d, J=9.1 Hz, 1H); 7.34 (dd, J=9.1, 2.7 Hz, 1H); 7.30 (d, J=2.7 Hz, 1H); 7.22-7.17 (m, 3H); 6.85 (apparent d, J=8.7 Hz, 2H); 4.46 (t, J=4.8 Hz, 1H); 4.19 (dd, J=10.9, 4.7 Hz, 2H); 3.94 (s, 3H); 3.79 (s, 3H); 3.74 (s, 2H); 3.28 (t, J=10.7 Hz, 2H); 3.11 (t, J=7.9 Hz, 2H); 13 3.05-2.94 (m, 1H); 2.08-2.01 (m, 2H);. C NMR (75 MHz, CDCl3) δ: 159.0, 157.8, 147.8, 146.2, 144.5, 132.3, 131.7, 129.3, 128.5, 121.5, 121.1, 114.1, 101.9, 101.0, 71.7, 55.6, 55.4,

+ 51.0, 49.9, 34.3, 26.4. HRMS (ESI) m/z calc’d for C24H29N2O4 [M+H] : 409.2127. found: 409.2098.

2-(2-(6-methoxyquinolin-4-yl)ethyl)-N-(4-(trifluoromethoxy)benzyl)-trans-1,3- dioxan-5-amine (30). The title compound was prepared in 62 % yield following the 1 general method and obtained as white solid. H NMR (300 MHz, CDCl3) δ: 8.67 (d, J=4.4 Hz, 1H); 8.02 (d, J=9.1 Hz, 1H); 7.40-7.29 (m, 4H); 7.23-7.15 (m, 3H); 4.48 (t, J=4.9 Hz, 1H); 4.23 (dd, J=11.2, 4.7 Hz, 2H); 3.95 (s, 3H); 3.83 (s, 2H); 3.31 (t, J=10.9 Hz, 2H); 3.14 13 (t, J=7.8 Hz, 2H); 3.06-2.96 (m, 1H); 2.13-2.02 (m, 2H). C NMR (75 MHz, CDCl3) δ: 157.8, 148.52, 148.50, 147.8, 146.2, 144.5, 139.0, 131.8, 129.3, 128.6, 122.3, 121.5, 121.2, 121.1, 118.9, 102.0, 101.1, 71.7, 55.6, 50.7, 50.0, 34.3, 26.4. HRMS (ESI) m/z calc’d for + C24H26F3N2O4 [M+H] : 463.1845. found: 463.1849.

N-(4-(tert-butyl)benzyl)-2-(2-(6-methoxyquinolin-4-yl)ethyl)-trans-1,3-dioxan-5- amine (33). The title compound was prepared in 60 % yield following the general method 1 and obtained as white solid. H NMR (300 MHz, CDCl3) δ: 8.66 (d, J=4.4 Hz, 1H); 8.01 (d, J=9.1 Hz, 1H); 7.35-7.40 (m, 3H); 7.30 (d, J=2.7 Hz, 1H); 7.23 (d, J=8.3 Hz, 2H); 7.18 (d, J=4.4 Hz, 1H); 4.47 (t, J=4.9 Hz, 1H); 4.22 (dd, J=11.1, 4.7 Hz, 2H); 3.94 (s, 3H); 3.78 (s, 2H); 3.30 (t, J=10.8 Hz, 2H); 3.13 (t, J=7.8 Hz, 2H); 3.07-2.97 (m, 1H); 2.13-2.00 (m, 13 2H); 1.31 (s, 9H). C NMR (75 MHz, CDCl3) δ: 157.8, 150.4, 147.8, 146.2, 144.5, 137.1, 131.7, 128.6, 127.8, 125.6, 121.5, 121.1, 101.9, 101.0, 71.7, 55.6, 51.1, 49.9, 34.6, 34.3, + 31.5, 26.5. HRMS (ESI) m/z calc’d for C27H35N2O3 [M+H] : 435.2648. found: 435.2646.

2-(2-(6-methoxyquinolin-4-yl)ethyl)-N-((5-methylpyridin-2-yl)methyl)-trans-1,3- dioxan-5-amine (34). The title compound was prepared in 73 % yield following the 1 general method and obtained as light brown solid. H NMR (300 MHz, CD3OD) δ: 8.54 (d, J=4.6 Hz, 1H); 8.39-8.32 (m, 1H); 7.94-7.86 (m, 1H); 7.65 (dd, J=7.9, 1.6 Hz, 1H); 7.43- 7.34(m, 3H); 7.32 (d, J=4.6 Hz, 1H); 4.56 (t, J=4.8 Hz, 1H); 4.22 (dd, J=11.3, 4.8 Hz, 2H); 3.94 (s, 3H); 3.92 (s, 2H); 3.43 (t, J=10.9 Hz, 2H); 3.15 (dd, J=9.0, 6.9 Hz, 2H); 3.04-2.91 13 (m, 1H); 2.35 (s, 3H); 2.08-1.96 (m, 2H). C NMR (75 MHz, CD3OD) δ: 159.6, 156.7, 150.0, 149.3, 148.2, 144.7, 139.4, 134.1, 131.2, 129.9, 123.7, 123.3, 122.4, 103.0, 102.2, + 71.4, 56.1, 52.0, 51.1, 35.4, 27.3, 18.0. HRMS (ESI) m/z calc’d for C23H28N3O3 [M+H] : 394.2130. found: 394.2121.

N-(4-(difluoromethoxy)benzyl)-2-(2-(6-methoxyquinolin-4-yl)ethyl)-trans-1,3- dioxan-5-amine (35). The title compound was prepared in 70 % yield following the 1 general method and obtained as white solid. H NMR (300 MHz, CDCl3) δ: 8.65 (d, J=4.4 Hz, 1H); 8.00 (d, J=9.1 Hz, 1H); 7.35 (dd, J=9.1, 2.7 Hz, 1H); 7.32-7.27 (m, 3H); 7.18 (d, J=4.4 Hz, 1H); 7.07 (d, J=8.5 Hz, 2H); 6.49 (t, J=74.0 Hz, 1H); 4.46 (t, J=4.9 Hz, 1H); 4.20 (dd, J=11.2, 4.7 Hz, 2H); 3.93 (s, 3H); 3.79 (s, 2H); 3.29 (t, J=10.9 Hz, 2H); 3.11 (t, 13 J=7.8 Hz, 2H); 3.03-2.93 (m, 1H); 2.12-1.98 (m, 2H). C NMR (75 MHz, CDCl3) δ: 157.8, 150.4, 147.8, 146.2, 144.5, 137.5, 131.7, 129.4, 128.5, 121.5, 121.1, 119.8, 119.5, 116.0, 112.6, 101.9, 101.0, 71.7, 55.6, 50.7, 50.0, 34.3, 26.4. HRMS (ESI) m/z calc’d for + C24H27F2N2O4 [M+H] : 445.1939. found: 445.1937.

N-(4-(difluoromethyl)benzyl)-2-(2-(6-methoxyquinolin-4-yl)ethyl)-trans-1,3-dioxan- 5-amine (36). The title compound was prepared in 49 % yield following the general 1 method and obtained as white solid. H NMR (300 MHz, CDCl3) δ: 8.65 (d, J=4.3 Hz, 1H); 8.01 (d, J=9.1 Hz, 1H); 7.44 (dd, J=21.7, 8.1 Hz, 4H); 7.36 (dd, J=9.2, 2.7 Hz, 1H); 7.29 (d, J=2.6 Hz, 1H); 7.19 (d, J=4.4 Hz, 1H); 6.63 (t, J=56.5 Hz, 1H); 4.47 (t, J=4.8 Hz, 1H); 4.21 (dd, J=11.1, 4.7 Hz, 2H); 3.94 (s, 3H); 3.86 (s, 2H); 3.30 (t, J=10.8 Hz, 2H); 3.12 (t, 13 J=7.8 Hz, 2H); 3.06-2.93 (m, 1H); 2.11-2.00 (m, 2H). C NMR (75 MHz, CDCl3) δ: 157.9, 147.8, 146.3, 144.5, 143.1, 133.9, 133.6, 133.3, 131.7, 128.6, 128.3, 126.0, 125.93, 125.85, 121.6, 121.1, 117.9, 114.8, 111.6, 102.0, 101.1, 71.6, 55.7, 51.1, 50.1, 34.3, 26.5. HRMS + (ESI) m/z calc’d for C24H27F2N2O3 [M+H] : 429.1990. found: 429.1988.

3-(((2-(2-(6-methoxyquinolin-4-yl)ethyl)-trans-1,3-dioxan-5- yl)amino)methyl)benzonitrile (37). The title compound was prepared in 58 % yield 1 following the general method and obtained as white solid. H NMR (300 MHz, CDCl3) δ: 8.66(d, J=4.5 Hz, 1H); 8.00 (d, J=9.1 Hz, 1H); 7.65 (s, 1H); 7.56-7.53 (m, 2H); 7.45-7.40 (m, 1H); 7.35 (dd, J=9.1, 2.7 Hz, 1H); 7.29 (d, J=2.7 Hz, 1H); 7.19 (d, J=4.5 Hz, 1H); 4.47 (t, J=4.9 Hz, 1H); 4.21 (dd, J=11.1, 4.9 Hz, 2H); 3.94 (s, 3H); 3.86 (s, 2H); 3.30 (t, J=10.9 Hz, 2H); 3.12 (t, J=7.9 Hz, 2H); 3.02-2.92 (m, 1H); 2.09-2.02 (m, 2H). 13C NMR (75 MHz,

CDCl3) δ: 157.8, 147.8, 146.2, 144.4, 141.9, 132.4, 131.7, 131.5, 131.1, 129.4, 128.5, 121.5, 121.1, 118.9, 112.7, 101.9, 101.1, 71.6, 55.7, 50.5, 50.1, 34.2, 26.4. HRMS (ESI) m/z calc’d + for C24H26N3O3 [M+H] : 404.1974. found: 404.1966.

N-(3-methoxybenzyl)-2-(2-(6-methoxyquinolin-4-yl)ethyl)-trans-1,3-dioxan-5-amine (38). The title compound was prepared in 60 % yield following the general method and 1 obtained as white solid. H NMR (300 MHz, CDCl3) δ: 8.66 (d, J=4.4 Hz, 1H); 8.01 (d, J=9.1 Hz, 1H); 7.35 (dd, J=9.1, 2.8 Hz, 1H); 7.29 (d, J=2.8 Hz, 1H); 7.24 (m, 1H); 7.19 (d, J=4.4 Hz, 1H); 6.89-6.86 (m, 2H); 6.82-6.78 (m, 1H); 4.46 (t, J=4.8 Hz, 1H); 4.21 (dd, J=11.0, 4.7 Hz, 2H); 3.93 (s, 3H); 3.80 (s, 3H); 3.79 (s, 2H); 3.30 (t, J=10.8 Hz, 2H); 3.12 (t, J=7.9 Hz, 2H); 3.06-2.96 (m, 1H); 2.09-2.02 (m, 2H). Solubility limitations precluded 13 + the acquisitions of a C NMR spectrum. HRMS (ESI) m/z calc’d for C24H29N2O4 [M+H] : 409.2127. found: 409.2092.

2-(2-(6-methoxyquinolin-4-yl)ethyl)-N-(2-methylbenzyl)-trans-1,3-dioxan-5-amine (40). The title compound was prepared in 65 % yield following the general method and 1 obtained as white solid. H NMR (300 MHz, CDCl3) δ: 8.66 (d, J=4.5 Hz, 1H); 8.01 (d, J=9.1 Hz, 1H); 7.35 (dd, J=9.1, 2.7 Hz, 1H); 7.30 (d, J=2.7 Hz, 1H); 7.27-7.23 (m, 1H); 7.20-7.15 (m, 4H); 4.48 (t, J=4.8 Hz, 1H); 4.24 (dd, J=11.1, 4.7 Hz, 2H); 3.94 (s, 3H); 3.80 (s, 2H); 3.31 (t, J=10.8 Hz, 2H); 3.13 (t, J=7.9 Hz, 2H); 3.08-2.99 (m, 1H); 2.34 (s, 3H); 13 2.10-2.03 (m, 2H). C NMR (75 MHz, CDCl3) δ: 157.8, 147.8, 146.2, 144.4, 138.0, 136.4, 131.7, 130.6, 128.5, 127.5, 126.2, 121.5, 121.1, 101.9, 101.0, 71.7, 55.6, 50.4, 49.4, 34.3, + 26.4, 19.0. HRMS (ESI) m/z calc’d for C24H29N2O3 [M+H] : 393.2178. found: 393.2148.

N-(2-methoxybenzyl)-2-(2-(6-methoxyquinolin-4-yl)ethyl)-trans-1,3-dioxan-5-amine (41). The title compound was prepared in 77 % yield following the general method and 1 obtained as white solid. H NMR (300 MHz, CDCl3) δ: 8.65(d, J=4.4 Hz, 1H); 8.00 (d, J=9.0 Hz, 1H); 7.35 (dd, J=9.0, 2.8 Hz, 1H); 7.30 (d, J=2.8 Hz, 1H); 7.28-7.21 (m, 2H); 7.18 (d, J=4.3 Hz, 1H); 6.95-6.85 (m, 2H); 4.46 (t, J=4.9 Hz, 1H); 4.18 (dd, J=11.0, 4.8 Hz, 2H); 3.93 (s, 3H); 3.84 (s, 3H); 3.80 (s, 2H); 3.29 (t, J=10.8 Hz, 2H); 3.12 (t, J=7.8 Hz, 13 2H); 3.03-2.93 (m, 1H); 2.08-2.02 (m, 2H). C NMR (75 MHz, CDCl3) δ: 157.8, 157.7, 147.9, 146.3, 144.5, 131.8, 129.9, 128.8, 128.6, 128.3, 121.6, 121.1, 120.8, 110.5, 101.9,

101.0, 71.8, 55.7, 55.4, 49.8, 47.3, 34.3, 26.5. HRMS (ESI) m/z calc’d for C24H29N2O4 [M+H]+: 409.2127. found: 409.2122.

N-(2-fluorobenzyl)-2-(2-(6-methoxyquinolin-4-yl)ethyl)-trans-1,3-dioxan-5-amine (43). The title compound was prepared in 70 % yield following the general method and 1 obtained as white solid. H NMR (300 MHz, CDCl3) δ: 8.65 (d, J=4.5 Hz, 1H); 8.00 (d, J=9.1 Hz, 1H); 7.36-7.28 (m, 3H); 7.26-7.21 (m, 1H); 7.17 (d, J=4.4 Hz, 1H); 7.13-7.07 (m, 1H); 7.06-7.00 (m, 1H); 4.46 (t, J=4.9 Hz, 1H); 4.20 (dd, J=11.1, 4.8 Hz, 2H); 3.93 (s, 3H); 3.85 (s, 2H); 3.29 (t, J=10.8 Hz, 2H); 3.11 (t, J=7.9 Hz, 2H); 3.04-2.94 (m, 1H); 2.08- 13 2.01 (m, 2H). C NMR (75 MHz, CDCl3) δ: 162.9, 159.6, 157.8, 147.8, 146.2, 144.5, 131.7, 130.33, 130.27, 129.2, 129.1, 128.5, 127.3, 127.1, 124.40, 124.35, 121.5, 121.1, 115.7, 115.4, 101.9, 101.0, 71.6, 55.6, 49.8, 45.13, 45.09, 34.3, 26.4. HRMS (ESI) m/z

+ calc’d for C23H26FN2O3 [M+H] : 397.1927. found: 397.1900 .

N-(3-chloro-4-fluorobenzyl)-2-(2-(6-methoxyquinolin-4-yl)ethyl)-trans-1,3-dioxan-5- amine (44). The title compound was prepared in 55 % yield following the general method 1 and obtained as white solid. H NMR (300 MHz, CDCl3) δ: 8.66(d, J=4.4 Hz, 1H); 8.01 (d, J=9.1 Hz, 1H); 7.39-7.33 (m, 2H); 7.29 (d, J=2.7 Hz, 1H); 7.19-7.13 (m, 2H); 7.07 (t, J=8.6 Hz, 1H); 4.46 (t, J=4.9 Hz, 1H); 4.20 (dd, J=11.0, 4.9 Hz, 2H); 3.93 (s, 3H); 3.76 (s, 2H); 3.28 (t, J=10.9 Hz, 2H); 3.12 (t, J=7.9 Hz, 2H); 3.02-2.91 (m, 1H); 2.08-2.02 (m, 2H);. 13C

NMR (75 MHz, CDCl3) δ: 159.1, 157.9, 155.8, 147.8, 146.2, 144.5, 137.41, 137.36, 131.8, 130.1, 128.6, 127.6, 127.5, 121.5, 121.2, 121.09, 120.98, 116.8, 116.5, 102.0, 101.1, 71.6, + 55.7, 50.3, 50.0, 34.3, 26.4. HRMS (ESI) m/z calc’d for C23H25ClFN2O3 [M+H] : 431.1538. found: 431.1515.

N-(4-chloro-3-fluorobenzyl)-2-(2-(6-methoxyquinolin-4-yl)ethyl)-trans-1,3-dioxan-5- amine (45). The title compound was prepared in 49 % yield following the general method 1 and obtained as white solid. H NMR (300 MHz, CDCl3) δ: 8.66(d, J=4.4 Hz, 1H); 8.01 (d, J=9.1 Hz, 1H); 7.38-7.29 (m, 3H); 7.19 (d, J=4.4 Hz, 1H); 7.14 (dd, J=9.8, 1.7 Hz, 1H); 7.03 (d, J=8.2 Hz, 1H); 4.46 (t, J=4.8 Hz, 1H); 4.20 (dd, J=11.0, 4.7 Hz, 2H); 3.94 (s, 3H); 3.79 (s, 2H); 3.29 (t, J=10.8 Hz, 2H); 3.12 (t, J=7.8 Hz, 2H); 3.02-2.91 (m, 1H); 2.09-2.03 13 (m, 2H);. C NMR (75 MHz, CDCl3) δ: 160.0, 157.9, 156.7, 147.8, 146.3, 144.5, 141.5, 141.4, 131.7, 130.7, 128.6, 124.22, 124.17, 121.6, 121.1, 119.8, 119.6, 116.2, 115.9, 102.0,

101.1, 71.6, 55.7, 50.4, 40.1, 34.3, 26.5. HRMS (ESI) m/z calc’d for C23H25ClFN2O3 96

[M+H]+: 431.1538. found: 431.1507.

N-(3,4-difluorobenzyl)-2-(2-(6-methoxyquinolin-4-yl)ethyl)-trans-1,3-dioxan-5-amine (46). The title compound was prepared in 44 % yield following the general method and 1 obtained as white solid. H NMR (300 MHz, CDCl3) δ: 8.66(d, J=4.5 Hz, 1H); 8.01 (d, J=9.1 Hz, 1H); 7.35(dd, J=9.1, 2.7 Hz, 1H); 7.29 (d, J=2.6 Hz, 1H); 7.19-7.01 (m, 4H); 4.46 (t, J=4.7 Hz, 1H); 4.20 (dd, J=11.0, 4.6 Hz, 2H); 3.93 (s, 3H); 3.77 (s, 2H); 3.28 (t, J=10.9 Hz, 2H); 3.12 (t, J=7.9 Hz, 2H); 3.01-2.91 (m, 1H); 2.09-2.02 (m, 2H). 13C NMR

(75 MHz, CDCl3) δ: 157.9, 152.2, 152.1, 151.4, 151.2, 148.9, 148.8, 148.1, 147.9, 147.8, 146.3, 144.4, 137.46, 137.39, 137.34, 131.7, 128.6, 123.78, 123.73, 123.70, 123.65, 121.6, 121.1, 117.4, 117.2, 116.9, 116.7, 102.0, 101.1, 71.6, 55.7, 50.4, 50.0, 34.3, 26.5. HRMS + (ESI) m/z calc’d for C23H25F2N2O3 [M+H] : 415.1833. found: 415.1814.

N-(3,5-difluorobenzyl)-2-(2-(6-methoxyquinolin-4-yl)ethyl)-trans-1,3-dioxan-5-amine (47). The title compound was prepared in 54 % yield following the general method and 1 obtained as white solid. H NMR (300 MHz, CDCl3) δ: 8.66 (d, J=4.4 Hz, 1H); 8.01 (d, J=9.1 Hz, 1H); 7.35 (dd, J=9.1, 2.7 Hz, 1H); 7.29 (d, J=2.7 Hz, 1H); 7.19 (d, J=4.4 Hz, 1H); 6.85 (d, J=7.0 Hz, 2H); 6.73-6.66 (m, 1H); 4.47 (t, J=4.8 Hz, 1H); 4.21 (dd, J=11.0, 4.7 Hz, 2H); 3.94 (s, 3H); 3.81 (s, 2H); 3.29 (t, J=10.8 Hz, 2H); 3.10 (t, J=7.9 Hz, 2H); 13 3.01-2.91 (m, 1H); 2.09-2.03 (m, 2H);. C NMR (75 MHz, CDCl3) δ: 165.0, 164.8, 161.7, 161.5, 157.9, 147.8, 146.2, 144.7, 144.58, 144.51, 144.47, 131.8, 128.6, 121.5, 121.1, 110.7, 110.6, 110.5, 110.4, 103.1, 102.7, 102.4, 101.9, 101.1, 71.6, 55.7, 50.6, 50.0, 34.3, 26.5. 97

+ HRMS (ESI) m/z calc’d for C23H25F2N2O3 [M+H] : 415.1833. found: 415.1809.

N-(2,3-difluorobenzyl)-2-(2-(6-methoxyquinolin-4-yl)ethyl)-trans-1,3-dioxan-5-amine (48). The title compound was prepared in 65 % yield following the general method and 1 obtained as white solid. H NMR (300 MHz, CDCl3) δ: 8.65(d, J=4.4 Hz, 1H); 8.01 (d, J=9.0 Hz, 1H); 7.35 (dd, J=9.0, 2.7 Hz, 1H); 7.29 (d, J=2.7 Hz, 1H); 7.18 (d, J=4.4 Hz, 1H); 7.13-7.00 (m, 3H); 4.46 (t, J=4.9 Hz, 1H); 4.20 (dd, J=11.0, 4.8 Hz, 2H); 3.94 (s, 3H); 3.88 (s, 2H); 3.30 (t, J=10.8 Hz, 2H); 3.12 (t, J=7.8 Hz, 2H); 3.03-2.93 (m, 1H); 2.09-2.02 13 (m, 2H). C NMR (75 MHz, CDCl3) δ: 157.9, 152.4, 152.2, 150.9, 150.7, 149.1, 148.9, 147.8, 147.6, 147.4, 146.2, 144.5, 131.8, 129.8, 129.7, 128.6, 124.85, 124.83, 124.39, 124.32, 124.23, 121.6, 121.1, 116.5, 116.3, 102.0, 101.1, 71.6, 55.7, 49.9, 44.7, 34.3, 26.5. + HRMS (ESI) m/z calc’d for C23H25F2N2O3 [M+H] : 415.1833. found: 415.1828.

N-(2,6-difluorobenzyl)-2-(2-(6-methoxyquinolin-4-yl)ethyl)-trans-1,3-dioxan-5-amine (49). The title compound was prepared in 76 % yield following the general method and 1 obtained as white solid. H NMR (300 MHz, CDCl3) δ: 8.65(d, J=4.5 Hz, 1H); 8.01 (d, J=9.1 Hz, 1H); 7.37-7.29 (m, 2H); 7.26-7.17 (m, 2H); 6.89 (t, J=7.8 Hz, 2H); 4.46 (t, J=4.8 Hz, 1H); 4.19 (dd, J=11.0, 4.7 Hz, 2H); 3.94 (s, 3H); 3.90 (s, 2H); 3.30 (t, J=10.8 Hz, 2H); 13 3.12 (t, J=7.9 Hz, 2H); 3.01-2.91 (m, 1H); 2.09-2.02 (m, 2H). C NMR (75 MHz, CDCl3) δ: 163.5, 163.4, 160.2, 160.1, 157.9, 147.8, 146.3, 144.5, 131.7, 129.5, 129.4, 129.2, 128.6,

121.6, 121.1, 116.2, 115.9, 115.7, 111.7, 111.6, 111.5, 111.4, 101.9, 101.1, 71.6, 55.7, 49.6, + 38.5, 34.3, 26.5. HRMS (ESI) m/z calc’d for C23H25F2N2O3 [M+H] : 415.1833. found: 415.1819.

N-(2,5-difluorobenzyl)-2-(2-(6-methoxyquinolin-4-yl)ethyl)-trans-1,3-dioxan-5-amine (50). The title compound was prepared in 56 % yield following the general method and 1 obtained as white solid. H NMR (300 MHz, CDCl3) δ: 8.66(d, J=4.5 Hz, 1H); 8.01 (d, J=9.1 Hz, 1H); 7.35(dd, J=9.1, 2.8 Hz, 1H); 7.29 (d, J=2.8 Hz, 1H); 7.18 (d, J=4.6 Hz, 1H); 7.10-7.05 (m, 1H); 7.03-6.88 (m, 2H); 4.47 (t, J=4.8 Hz, 1H); 4.21 (dd, J=11.0, 4.8 Hz, 2H); 3.94 (s, 3H); 3.84 (s, 2H); 3.30 (t, J=10.9 Hz, 2H); 3.12 (t, J=7.9 Hz, 2H); 3.03-2.93 13 (m, 1H); 2.09-2.03 (m, 2H). C NMR (75 MHz, CDCl3) δ: 160.5, 158.6, 157.9, 157.3, 155.4, 147.8, 146.2, 144.5, 131.8, 129.2, 129.1, 128.99, 128.89, 128.6, 121.6, 121.1, 116.7, 116.63, 116.59, 116.53, 116.41, 116.30, 116.27, 116.20, 115.5, 115.4, 115.2, 115.1, 102.0,

101.1, 71.6, 55.7, 49.9, 44.6, 34.3, 26.5. HRMS (ESI) m/z calc’d for C23H25F2N2O3 [M+H]+: 415.1833. found: 415.1799.

N-((6-methoxypyridin-3-yl)methyl)-2-(2-(6-methoxyquinolin-4-yl)ethyl)-trans-1,3- dioxan-5-amine (51). The title compound was prepared in 65 % yield following the 1 general method and obtained as light yellow solid. H NMR (300 MHz, CDCl3) δ: 8.65 (d, J=4.4 Hz, 1H); 8.06 (d, J=2.2 Hz, 1H); 8.00 (d, J=9.0 Hz, 1H); 7.54 (dd, J=8.6, 2.4 Hz, 1H); 7.35 (dd, J=9.1, 2.7 Hz, 1H); 7.29 (d, J=2.7 Hz, 1H); 7.19 (t, J=4.5 Hz, 1H); 6.71 (d, J=8.4 Hz, 1H); 4.45 (t, J=4.8 Hz, 1H); 4.20 (dd, J=11.1, 4.7 Hz, 2H); 3.93 (s, 3H); 3.92 (s, 99

3H); 3.73 (s, 2H); 3.28 (t, J=10.8 Hz, 2H); 3.11 (t, J=7.8 Hz, 2H); 3.03-2.93 (m, 1H); 2.09- 13 2.02 (m, 2H). C NMR (75 MHz, CDCl3) δ: 163.8, 157.8, 147.8, 146.2, 144.5, 138.9, 131.8, 128.6, 128.3, 121.5, 121.1, 111.0, 101.9, 101.0, 71.7, 55.7, 53.6, 49.8, 48.3, 34.3, + 26.4. HRMS (ESI) m/z calc’d for C23H28N3O4 [M+H] : 410.2080. found: 410.2067.

N-((6-methoxypyridin-3-yl)methyl)-2-(2-(6-methoxyquinolin-4-yl)ethyl)-trans-1,3- dioxan-5-amine (52). The title compound was prepared in 59 % yield following the 1 general method and obtained as light yellow solid. H NMR (300 MHz, CDCl3) δ: 8.66 (d, J=4.4 Hz, 1H); 8.19 (d, J=18.3 Hz, 2H); 8.01 (d, J=9.2 Hz, 1H); 7.36 (dd, J=9.1, 2.7 Hz, 1H); 7.29 (d, J=2.7 Hz, 1H); 7.19 (t, J=4.3 Hz, 2H); 4.47 (t, J=4.9 Hz, 1H); 4.23 (dd, J=11.2, 4.7 Hz, 2H); 3.94 (s, 3H); 3.86 (s, 3H); 3.83 (s, 2H); 3.30 (t, J=10.8 Hz, 2H); 3.15 (t, J=7.8 13 Hz, 2H); 3.04-2.94 (m, 1H); 2.13-2.00 (m, 2H). C NMR (75 MHz, CDCl3) δ: 157.9, 147.7, 146.4, 144.4, 141.8, 136.7, 131.7, 128.6, 121.6, 121.1, 120.2, 101.9, 101.0, 71.6, 55.7, 50.1, + 48.6, 34.2, 26.4. HRMS (ESI) m/z calc’d for C23H28N3O4 [M+H] : 410.2080. found: 410.2076.

2-(2-(6-methoxyquinolin-4-yl)ethyl)-N-(thiophen-3-ylmethyl)-trans-1,3-dioxan-5- amine (53). The title compound was prepared in 69 % yield following the general method 1 and obtained as white solid. H NMR (300 MHz, CDCl3) δ: 8.65(d, J=4.4 Hz, 1H); 8.00(d, J=9.1 Hz, 1H); 7.34(dd, J=9.1, 2.7 Hz, 1H); 7.31-7.25 (m, 2H); 7.17 (d, J=4.4 Hz, 1H); 7.12(d, J=1.8 Hz, 1H); 7.02 (dd, J=4.9, 1.1 Hz, 1H); 4.46 (t, J=4.9 Hz, 1H); 4.19 (dd, J=11.1, 4.7 Hz, 2H); 3.93 (s, 3H); 3.83 (s, 2H); 3.28 (t, J=10.8 Hz, 2H); 3.12 (t, J=7.9 Hz,

100

13 2H); 3.06-2.96 (m, 1H); 2.00-2.10 (m, 2H). C NMR (75 MHz, CDCl3) δ: 157.8, 147.8, 146.2, 144.5, 141.3, 131.7, 128.5, 127.4, 126.2, 121.8, 121.5, 121.0, 101.9, 101.0, 71.6, + 55.6, 50.0, 46.5, 34.3, 26.4. HRMS (ESI) m/z calc’d for C21H24N2O3SNa [M+Na] : 407.1405. found: 407.1374.

N-((1H-indol-7-yl)methyl)-2-(2-(6-methoxyquinolin-4-yl)ethyl)-trans-1,3-dioxan-5- amine (54). The title compound was prepared in 39 % yield following the general method 1 and obtained as light yellow solid. H NMR (300 MHz, CDCl3) δ: 9.37 (s, 1H); 8.66(d, J=4.4 Hz, 1H); 8.01(d, J=9.2 Hz, 1H); 7.58 (d, J=7.5 Hz, 1H); 7.36(dd, J=9.1, 2.7 Hz, 1H); 7.29 (d, J=2.6 Hz, 1H); 7.24-7.20 (m, 1H); 7.19 (d, J=4.4 Hz, 1H); 7.09-6.95 (m, 2H); 6.59-6.50 (m, 1H); 4.47 (t, J=4.8 Hz, 1H); 4.27 (dd, J=11.0, 4.7 Hz, 2H); 4.19 (s, 2H); 3.93 (s, 3H); 3.32 (t, J=10.8 Hz, 2H); 3.14-3.01 (m, 3H); 2.13-1.99 (m, 2H). 13C NMR (75 MHz,

CDCl3) δ: 157.9, 147.8, 146.2, 144.5, 135.4, 131.8, 128.6, 128.4, 124.0, 121.9, 121.6, 121.10, 121.05, 120.2, 119.6, 102.5, 101.9, 101.0, 71.5, 55.7, 50.6, 50.0, 34.2, 26.4. HRMS + (ESI) m/z calc’d for C25H28N3O3 [M+H] : 418.2131. found: 418.2136.

N-((6,7-dihydro-[1,4]oxathiino[2,3-c]pyridazin-3-yl)methyl)-2-(2-(6- methoxyquinolin-4-yl)ethyl)-trans-1,3-dioxan-5-amine (55). The title compound was prepared in 39 % yield following the general method and obtained as yellow solid. 1H NMR

(300 MHz, CDCl3) δ: 8.65(d, J=4.5 Hz, 1H); 8.03(d, J=9.2 Hz, 1H); 7.36(dd, J=9.2, 2.7

101

Hz, 1H); 7.30 (d, J=2.7 Hz, 1H); 7.27 (s, 1H); 7.20 (d, J=4.5 Hz, 1H); 4.64(t, J=4.7 Hz, 2H); 4.48 (t, J=4.8 Hz, 1H); 4.24 (dd, J=11.2, 4.7 Hz, 2H); 3.97 (s, 2H); 3.94 (s, 3H); 3.33 (t, J=10.9 Hz, 2H); 3.20 (t, J=4.7 Hz, 2H); 3.13 (t, J=7.9 Hz, 2H); 3.07-2.93 (m, 1H); 2.12- 13 1.97 (m, 2H). C NMR (75 MHz, CDCl3) δ: 160.1, 158.0, 156.3, 147.3, 146.9, 143.9, 131.3, 128.6, 126.0, 125.2, 121.9, 121.1, 101.9, 101.0, 71.4, 66.3, 55.7, 50.2, 50.2, 34.3, + 26.5, 25.7. HRMS (ESI) m/z calc’d for C23H27N4O4S [M+H] : 455.1753. found: 455.1753.

2-(hydroxymethyl)-5-((4-methoxybenzyl)oxy)-4H-pyran-4-one (59). Kojic acid 58 (5.0 g, 35.3 mmol, 1.0 equiv) and K2CO3 (10.0 g, 72.3 mmol, 2.05 equiv) were dispersed in dry N,N-dimethylformamide (45 mL) with Argon protection. 4-Methoxybenzyl chloride (5.0 mL, 37 mmol, 1.05 equiv) was dropped slowly at 0 °C. The mixture was heated to 85 °C and stirred for 2h. Then the reaction was quenched by addition of H2O (5 mL), and the solvent was removed under vacuum. The residue was mixed with ice H2O (200 mL) and the precipate was collected via filtration. The filtrate was washed by H2O (50 mL) three times, and then by icy ethyl acetate (75 mL). In the end, the waste ethyl acetate was colorless. The title compound was obtained in ca. 95% purity as a light brown solid (7.3 g, 1 27.8 mmol, 79%) after vacuum drying. H NMR (300 MHz, DMSO-d6) δ: 8.12 (s, 1H); 7.32 (d, J = 8.7, 2H); 6.93 (d, J = 8.7, 2H); 6.30 (s, 1H); 5.66 (s, 1H); 4.85 (s, 2H); 4.28 (s, 2H); 3.75 (s, 3H).

2-(hydroxymethyl)-5-((4-methoxybenzyl)oxy)pyridin-4(1H)-one (60). Intermediate 59 (7.3 g, 27.8 mmol, 1.0 equiv) was dissolved in the mixture of EtOH (30 mL) and ammonium hydroxide (30 mL). The reaction mixture was stirred at 45 °C for 1d, and then cooled down to 0 °C. The white precipate was collected via filtration, then washed by ice- 102 water (15 mL) 3 times and icy EtOH (8 mL) 3 times. The title compound was obtained as a white solid (3.8 g, 14.5 mmol, 52%) after drying.

(5-((4-methoxybenzyl)oxy)-4-oxo-1,4-dihydropyridin-2-yl)methyl acetate (61). Intermediate 60 (3.8 g, 14.5 mmol, 1.0 equiv) was dissolved in pyridine (55 mL), acetyl chloride (1.27 mL, 17.4 mmol, 1.5 equiv) was dropped at 0 °C. The reaction mixture was stirred at room temperature for 12h, and then stirred at 85 °C for 2h. Purification by chromatography on silica gel with DCM/methanol (15:1) afforded the title compound as a white solid (3.254 g, 10.73 mmol, 74%).

(5-((4-methoxybenzyl)oxy)-4-(((trifluoromethyl)sulfonyl)oxy)pyridin-2-yl)methyl acetate (62). Intermediate 61 (3.254 g, 10.73 mmol, 1.0 equiv) and triethylamine (4.5 mL, 32.1 mmol, 3.0 equiv) were dissolved in DCM (50 mL), trifluoromethanesulfonic anhydride (3.605 mL, 21.46 mmol, 2.0 equiv) was added slowly at 0 °C. The reaction mixture was stirred at room temperature for 36h. Then the solvent was removed and the crude product was purified by chromatography on silica gel with hexane/ethyl acetate (3:1). The title compound was obtained as yellow oil (3.553 g, 8.16mmol, 76%). 1H NMR (300

MHz, CDCl3) δ: 8.44 (s, 1H); 7.35 (d, J=8.7 Hz, 2H); 7.25 (s, 1H); 6.90 (d, J=8.7 Hz, 2H); 5.18 (s, 2H); 5.14 (s, 2H); 3.79 (s, 3H); 2.13 (s, 3H).

(4-(3-hydroxyprop-1-yn-1-yl)-5-((4-methoxybenzyl)oxy)pyridin-2-yl)methyl acetate (63). Intermediate 62 (3.553 g, 8.16 mmol, 1.0 equiv), copper(I) iodide (154.7 mg, 0.816

103 mmol, 0.1 equiv), PdCl2(PPh3)2 (290.4 mg, 0.408 mmol, 0.05 equiv) and propargyl alcohol (1.466 mL, 24.48 mmol, 3 equiv) were placed in a round flask with argon protection. Degassed acetonitrile (50 mL) was added. The reaction mixture was stirred at 50 °C overnight. Then the solvent was removed and the crude product was purified by chromatography on silica gel with hexane/ethyl acetate (1:1). The title compound was 1 obtained as brown oil (2.618 g, 7.67 mmol, 94%). H NMR (300 MHz, CDCl3) δ: 8.27 (s, 1H); 7.33 (d, J=8.7 Hz, 2H); 7.32 (s, 1H); 6.89 (d, J=8.7 Hz, 2H); 5.13 (s, 2H); 5.10 (s, 2H); 4.51 (s, 2H); 3.79 (s, 3H); 2.46 (brs, 1H); 2.12 (s, 3H).

(5-hydroxy-4-(3-hydroxypropyl)pyridin-2-yl)methyl acetate (64). Intermediate 63 (2.618 g, 7.67 mmol, 1.0 equiv) and 10wt% Pd/C (404.4 mg, 0.38 mmol, 0.05 equiv) were dispersed in EtOH (30 mL). The mixture was degased with H2, and then stirred with a H2 balloon overnight. The title compound was obtained as a white solid (1.313 g, 5.83 mmol, 76%) after purification by chromatography on silica gel with DCM/methanol (15:1).

(3,4-dihydro-2H-pyrano[2,3-c]pyridin-6-yl)methanol (66). Diisopropyl azodicarboxylate (1.72 mL, 8.75 mmol, 1.5 equiv) and PPh3 (2.295 g, 8.75 mmol, 1.5 equiv) were dissolved in THF (30 mL) and stirred for 1h. Intermediate 64 (1.313 g, 5.83 mmol, 1.0 equiv) in THF (45 mL) solution was added slowly. Then the mixture was stirred overnight. The solvent was removed by vacuum, and the organics were washed by brine and extracted by ethyl acetate. Crude product (containing intermediate 65) was obtained after drying and added into 2M NaOH solution (40 mL) directly. After the completion of hydrolysis, pH value of the reaction mixture was adjusted to 7 by 1M HCl solution. Then the mixture was extracted by ethyl acetate and purified by chromatography on silica gel with DCM/methanol (15:1). White crystal (757.4 mg, 4.58 mmol, 79%) was obtained as 104

1 the title compound. H NMR (300 MHz, CDCl3) δ: 8.02 (s, 1H); 6.95 (s, 1H); 4.60 (s, 2H); 4.18 (t, J=5.2 Hz, 2H); 4.05 (brs, 1H); 2.74 (t, J=6.5 Hz, 2H); 2.04-1.93 (m, 2H).

3,4-dihydro-2H-pyrano[2,3-c]pyridine-6-carbaldehyde (67). Intermediate 66 (757.4 mg, 4.58 mmol, 1.0 equiv) was dissolved in DCM (30 mL). Dess-Martin periodinane (2002.6 mg, 4.58 mmol, 1.0 equiv) was added slowly. The mixture was stirred for 30min, and then saturated Na2S2O3 aqueous solution (20 mL) and saturated NaHCO3 aqueous solution (20 mL) were added. The organic layer became transparent after 3h, and then the organics were collected and extracted by DCM. Purification by chromatography on silica gel with hexane/ethyl acetate (4:1) afforded the title compound as a white solid (642.7 mg, 3.94 1 mmol, 86%). H NMR (300 MHz, CDCl3) δ: 9.92 (s, 1H); 8.24 (s, 1H); 7.69 (s, 1H); 4.30 (t, J=5.2 Hz, 2H); 2.83 (t, J=6.4 Hz, 2H); 2.09-2.02 (m, 2H).

methyl 6-amino-5-bromopicolinate (69). Compound 68 (5.9 g, 38.8 mmol, 1.0 equiv) was dissolved in chloroform (60 mL), N-bromosuccinimide (7.25 g, 40.74 mmol, 1.05 equiv) was added. The reaction mixture was stirred overnight. Then the solvent was removed and the crude product was purified by chromatography on silica gel with hexane/ethyl acetate (3:1). The title compound was obtained as a yellow solid (1.7 g, 7.37 1 mmol, 19%). H NMR (300 MHz, CDCl3) δ: 7.77 (d, J=7.9 Hz, 1H); 7.35 (d, J=7.9 Hz, 1H); 5.32 (brs, 2H); 3.95 (s, 3H).

105 methyl 3-oxo-3,4-dihydro-2H-pyrido[3,2-b][1,4]thiazine-6-carboxylate (70). Ethyl 2- mercaptoacetate (835 L, 7.46 mmol, 1.01 equiv) was dissolved in anhydrous DMF (20 mL) under Argon protection, and then 60% NaH powder (306 mg, 7.65 mmol, 1.04 equiv) was added at 0 °C. The reaction mixture was stirred at 0 °C for 1h, and then intermediate 69 (1.7 g, 7.37 mmol, 1.0 equiv) in anhydrous DMF (8 mL) solution was dropwised. The reaction mixture was stirred at room temperature for 16h. The mixture was diluted with ethyl acetate (566 mL) and washed by pure H2O (165 mL) 3 times. The organic solution was concentrated to the volume of 5 mL, and then white precipitate was collected and washed by ethyl acetate (2 mL) twice to give the title compound (609 mg, 2.72 mmol, 37%).

3-oxo-3,4-dihydro-2H-pyrido[3,2-b][1,4]thiazine-6-carboxylic acid (71). To a dioxane/H2O (92 mL/23 mL) solution of intermediate 70 (609 mg, 2.72 mmol, 1.0 equiv), 0.5 M NaOH aqueous solution (6.2 mL) was dropped over 2h at room temperature. The reaction mixture was stirred overnight. Then the volume of the mixture was concentrated to 2 mL. Some extra H2O (4 mL) was added and pH value of the mixture was adjusted to 4 with 1M HCl. After that, the precipate was collected and washed by a small amount of

H2O (5 mL). A white solid (300.5 mg, 1.43 mmol, 53%) was obtained after drying as the 1 title compound. H NMR (300 MHz, MeOD-d4) δ: 7.89 (d, J=7.9 Hz, 1H); 7.76 (d, J=7.9 Hz, 1H); 3.61 (s, 2H).

6-(hydroxymethyl)-2H-pyrido[3,2-b][1,4]thiazin-3(4H)-one (73). To a THF (14 mL) solution of intermediate 71 (300.5 mg, 1.43 mmol, 1.0 equiv) and triethylamine (238 L, 1.71 mmol, 1.2 equiv), isobutyl chloroformate (204 L, 1.57 mmol, 1.1 equiv ) was

106 dropped at -10 °C. The mixture was stirred for 20min, and then it was filtered through a glass funnel with silica gel (10 g) and directly into an ice-cooled H2O (4.8 mL) solution of

NaBH4 (163.2 mg, 4.31 mmol, 3.0 equiv). The reaction mixture was stirred at 0 °C for 30min. Then pH value of the mixture was adjusted to 7 with 1M HCl. The solvent was removed in vacuum, and then white solid was washed by pure H2O (5 mL) to give the title 1 compound (160 mg, 0.81 mmol, 58%). H NMR (300 MHz, DMSO-d6) δ: 10.84 (s, 1H); 7.76 (d, J=7.9 Hz, 1H); 7.09 (d, J=7.9 Hz, 1H); 5.40 (t, J=5.8 Hz, 1H); 4.45 (d, J=5.8 Hz, 2H); 3.52 (s, 2H).

3-oxo-3,4-dihydro-2H-pyrido[3,2-b][1,4]thiazine-6-carbaldehyde (74). Intermediate 73 (160 mg, 0.81 mmol, 1.0 equiv) was dissolved in DCM (4 mL). Dess-Martin periodinane (376 mg, 0.82 mmol, 1.01 equiv) was added slowly. The mixture was stirred for 30min, and then saturated Na2S2O3 aqueous solution (4 mL) and saturated NaHCO3 aqueous solution (4 mL) were added. The organic layer became transparent after 3h, and then the organics were collected and extracted by DCM. Purification by chromatography on silica gel with DCM/methanol (15:1) afforded the title compound as a light yeloow solid 1 (122 mg, 0.682 mmol, 77%). H NMR (300 MHz, CDCl3) δ: 9.91 (s, 1H); 8.45 (brs, 1H); 7.80 (d, J=7.9 Hz, 1H); 7.62 (d, J=7.9 Hz, 1H); 3.59 (s, 2H).

6-bromo-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one (78). To a solution of NaOMe (567.2 mg, 10.5 mmol, 1.05 equiv) in MeOH (25 mL) was added 3-hydroxy-2-nitropyridine 75 (1.4 g, 10.0 mmol, 1.0 equiv) at room temperature. Bromine (514 L, 10.0 mmol, 1.0 equiv) was dropped at 0°C, and then the mixture was stirred at 0°C for 2h. The reaction was quenched by addition of acetic acid (180 L), then crude intermediate 76 was obtained via drying and used directly. To the suspension of crude intermediate 76 and K2CO3 (2.77 g, 107

20 mmol, 2.0 equiv) in aceton (15 mL) was added ethyl bromoacetate (1.11 mL, 10 mmol, 1.0 equiv), and then the mixture was stirred at 57°C for 8h. After dilution of the mixture with t-butyl methyl ether (14 mL), the resulting precipitate was filtered off. The filtrate was collected and concentrated to dryness which afforded crude intermediate 77. The crude intermediate 77 was dissolved in acetic acid (12 mL) and iron powder (1.62 g, 29 mmol) was added. The mixture was stirred at 90°C for 3.5h and then diluted with ethyl acetate (24 mL). The resulting precipitate was filtered off and washed by ethyl acetate (10 mL) three times. The filtrate was collected and concentrated in vacuum. Purification by chromatography on silica gel with hexane/ethyl acetate (3:1) afforded the title compound 1 as a light yellow solid (364.3 mg, 1.59 mmol, 16% in 3 steps). H NMR (300 MHz, CDCl3) δ: 8.28 (brs, 1H); 7.14 (d, J=8.2 Hz, 1H); 7.09 (d, J=8.2 Hz, 1H); 4.67 (s, 2H).

(E)-6-styryl-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one (79). In a three-necked flask intermediate 78 (364.3 mg, 1.59 mmol, 1.0 equiv), trans-2-phenylvinylboronic acid (423.5 mg, 2.86 mmol, 1.8 equiv), K2CO3 (450 mg, 3.18 mmol, 2.0 equiv) and Pd(PPh3)4 (92.7 mg, 0.0795 mmol, 0.05 equiv) were placed with argon protection. 1,4-dioxane (6.3 mL) and H2O (1 mL) were added. The reaction mixture was stirred at 100°C for 1d. Then the mixture was purified via chromatography on silica gel with hexane/ethyl acetate (3:1) to give the title compound as a white solid (168 mg, 0.67 mmol, 42%). 1H NMR (300 MHz,

CDCl3) δ: 8.20 (brs, 1H); 7.58-7.50 (m, 2H); 7.46 (d, J=16.0 Hz, 1H); 7.41-7.32 (m, 2H); 7.32-7.27 (m, 1H); 7.23 (d, J=8.2 Hz, 1H); 7.04 (d, J=7.9 Hz, 1H); 7.02 (d, J=8.2 Hz, 1H); 4.69 (s, 2H).

3-oxo-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazine-6-carbaldehyde (80). Intermediate 79 (168 mg, 0.67mmol, 1.0 equiv) was dissolved in the mixture of 1,4-dioxane (2.0 mL) and

108

H2O (0.5 mL). To the reaction mixture, 5.0 wt.% solution of OsO4 in tert-butanol (35 L,

0.0067mmol, 0.01 equiv) was added. After 20 min, NaIO4 (427.2 mg, 2.0 mmol, 3.0 equiv) was added. The mixture was stirred for 1d. Then the solvent was removed in vacuum and purification by chromatography on silica gel with hexane/ethyl acetate (3:1) afforded the 1 title compound as a brown solid (59 mg, 0.33mmol, 50%). H NMR (300 MHz, CDCl3) δ: 9.88 (s, 1H); 8.28 (brs, 1H); 7.68 (d, J=8.1 Hz, 1H); 7.38 (d, J=8.1 Hz, 1H); 4.79 (s, 2H).

3-(6-methoxyquinolin-4-yl)prop-2-yn-1-ol (81). In a three-necked flask intermediate 2

(715 mg, 3.0 mmol, 1.0 equiv), propargyl alcohol (528L, 9.0 mmol, 3.0 equiv), CuI2 (57.3 mg, 0.3 mmol, 0.1 equiv), PdCl2(PPh3)2 (107.7 mg, 0.15 mmol, 0.05 equiv) and triethylamine (2.09 mL, 15 mmol, 5.0 equiv) were placed with argon protection. CH3CN (25 mL) was added. The reaction mixture was stirred at 50°C overnight. Then the mixture was purified by chromatography on silica gel with hexane/ethyl acetate (1:1) and afforded the title compound as a light yellow solid (486 mg, 2.27 mmol, 76%).

3-(6-methoxyquinolin-4-yl)propan-1-ol (82). Intermediate 81 ((486 mg, 2.27 mmol, 1.0 equiv) and 10wt% Pd/C (120.8 mg, 0.114 mmol, 0.05 equiv) were dispersed in methanol

(12 mL). The mixture was stirred in H2 atmosphere overnight. Purification by chromatography on silica gel with DCM/methanol (30:1) afforded the title compound as a 1 yellow solid (468.4 mg, 2.16 mmol, 95%). H NMR (300 MHz, CDCl3) δ: 8.59 (d, J=4.5 Hz, 1H); 7.98 (d, J=9.1 Hz, 1H); 7.33 (dd, J=9.1, 2.8 Hz, 1H); 7.27 (d, J=2.7 Hz, 1H); 7.17

109

(d, J=4.5 Hz, 1H); 3.90 (s, 3H); 3.76 (t, J=6.2 Hz, 2H); 3.16-3.08 (m, 2H); 2.90 (brs, 1H); 2.09-1.96 (m, 2H).

3-(6-methoxyquinolin-4-yl)acrylaldehyde (83). Intermediate 82 (468.4 mg, 2.16 mmol, 1.0 equiv) was dissolved in DCM (25 mL). Dess-Martin periodinane (1133 mg, 2.59 mmol, 1.2 equiv) was added slowly. The mixture was stirred overnight, and then saturated

Na2S2O3 aqueous solution (20 mL) and saturated NaHCO3 aqueous solution (20 mL) were added. The organic layer became transparent after 3h, and then the organics were collected and extracted by DCM. Purification by chromatography on silica gel with DCM/methanol (50:1) afforded the title compound as a white solid (282 mg, 1.32 mmol, 61%). 1H NMR

(300 MHz, CDCl3) δ: 9.77 (d, J=8.1 Hz, 1H); 8.80 (d, J=4.4 Hz, 1H); 8.08 (d, J=9.2 Hz, 1H); 8.03 (dd, J=11.6, 0.7 Hz, 1H); 7.43 (dd, J=9.2, 2.8 Hz, 1H); 7.30 (dd, J=4.4, 0.9 Hz, 1H); 7.09 (d, J=2.7 Hz, 1H); 6.51 (dd, J=11.6, 8.1 Hz, 1H); 3.94 (s, 3H).

2-(2-((E)-2-(6-methoxyquinolin-4-yl)vinyl)-trans-1,3-dioxan-5-yl)isoindoline-1,3- dione (84). Intermediate 83 (282 mg, 1.32 mmol, 1.0 equiv) and Intermediate 5 (350.4 mg, 1.58 mmol, 1.2 equiv) were dispersed in toluene (40 mL). p-toluenesulfonic acid ( 11.4 mg, 0.066mmol, 0.05equiv) was added. The reaction mixture was stirred at 110°C for 2d. 110

Purification by chromatography on silica gel with DCM/methanol (50:1) afforded the title 1 compound as a white solid (325 mg, 0.78 mmol, 59%). H NMR (300 MHz, CDCl3) δ: 8.74 (d, J=4.6 Hz, 1H); 8.03 (d, J=9.2 Hz, 1H); 7.88-7.84 (m, 2H); 7.80-7.72 (m, 2H); 7.48 (d, J=13.4 Hz, 1H); 7.46-7.44 (m, 1H); 7.39 (dd, J=9.2, 2.7 Hz, 1H); 7.30 (d, J=2.6Hz, 1H); 6.42 (dd, J=15.9, 4.2 Hz, 1H); 5.42 (d, J=3.8 Hz, 1H); 4.82-4.71 (m, 1H); 4.62 (t, J=10.2 13 Hz, 2H); 4.18 (dd, J=10.4, 4.6 Hz, 2H); 3.98 (s, 3H). C NMR (75 MHz, CDCl3) δ: 168.0, 158.1, 147.7, 144.8, 140.6, 134.6, 131.7, 131.6, 131.0, 128.9, 127.4, 123.7, 122.2, 118.4, + 101.7, 99.9, 66.7, 55.8, 44.2. HRMS (ESI) m/z calc’d for C24H21N2O5 [M+H] : 417.1450. found: 417.1439.

2-((E)-2-(6-methoxyquinolin-4-yl)vinyl)-trans-1,3-dioxan-5-amine (85). A mixture of compound 84 (320 mg, 0.77 mmol, 1.0 equiv), ethanolamine (927 L, 15.36 mmol, 20 equiv) and ethyl acetate (20 mL) was stirred and heated at 70 °C for 3d. The solvent was removed, and the mixture was dissolved in DCM and washed with brine. The organic layer was combined and concentrated, and the crude product was purified by chromatography on silica gel with DCM/methanol (15:1) to give the title compound as yellow oil (162 mg, 1 0.57 mmol, 73%). H NMR (300 MHz, CDCl3) δ: 8.71 (d, J=4.6 Hz, 1H); 8.00 (d, J=9.2 Hz, 1H); 7.43 (d, J=6.6 Hz, 1H); 7.39 (d, J=4.1 Hz, 1H); 7.37 (dd, J=9.2, 2.7 Hz, 1H); 7.28 (d, J=2.7Hz, 1H); 6.37 (dd, J=16.0, 4.1 Hz, 1H); 5.14 (d, J=4.1 Hz, 1H); 4.27 (dd, J=10.6, 4.9 Hz, 2H); 3.95 (s, 3H); 3.41 (t, J=10.2 Hz, 2H); 3.26-3.11 (m, 1H);. 13C NMR (75 MHz,

CDCl3) δ: 158.0, 147.8, 144.9, 140.6, 131.6, 131.3, 128.6, 127.4, 122.0, 118.4, 101.8, 99.5, + 73.7, 55.8, 44.3. HRMS (ESI) m/z calc’d for C16H19N2O3 [M+H] : 287.1396. found: 111

287.1374.

N-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-2-((E)-2-(6-methoxyquinolin-4- yl)vinyl)-trans-1,3-dioxan-5-amine (86). The title compound was prepared in 74 % yield following the general reductive amination procedure and obtained as white solid. 1H NMR

(300 MHz, CDCl3) δ: 8.70 (d, J=4.6 Hz, 1H); 7.99 (d, J=9.2 Hz, 1H); 7.41 (d, J=4.9 Hz, 1H); 7.39-7.33 (m, 2H); 7.27 (d, J=2.7 Hz, 1H); 6.89-6.73 (m, 3H); 6.35 (dd, J=16.0, 4.1 Hz, 1H); 5.14 (d, J=4.0 Hz, 1H); 4.29 (dd, J=11.2, 4.7 Hz, 2H); 4.23 (s, 4H); 3.94 (s, 3H); 13 3.72 (s, 2H); 3.46 (t, J=10.8 Hz, 2H); 3.15-3.00 (m, 1H);. C NMR (75 MHz, CDCl3) δ: 158.0, 147.8, 144.8, 143.7, 142.9, 140.6, 133.5, 131.6, 131.4, 128.5, 127.4, 122.0, 121.1, 118.3, 117.4, 116.9, 101.8, 99.8, 71.8, 64.5, 64.5, 55.8, 50.9, 49.7. HRMS (ESI) m/z calc’d + for C25H27N2O5 [M+H] : 435.1920. found: 435.1908.

N-(3,4-dichlorobenzyl)-2-((E)-2-(6-methoxyquinolin-4-yl)vinyl)-trans-1,3-dioxan-5- amine (87). The title compound was prepared in 35 % yield following the general reductive

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1 amination procedure and obtained as white solid. H NMR (300 MHz, CDCl3) δ: 8.71 (d, J=4.6 Hz, 1H); 8.01 (d, J=9.2 Hz, 1H); 7.46-7.35 (m, 5H); 7.27 (d, J=2.7 Hz, 1H); 7.16 (dd, J=8.2, 2.0 Hz, 1H); 6.36 (dd, J=16.0, 4.1 Hz, 1H); 5.16 (d, J=4.1 Hz, 1H); 4.31 (dd, J=11.2, 4.7 Hz, 2H); 3.95 (s, 3H); 3.81 (s, 2H); 3.48 (t, J=10.9 Hz, 2H); 3.11-3.00 (m, 1H);. 13 C NMR (75 MHz, CDCl3) δ: 158.0, 147.7, 144.8, 140.7, 140.5, 132.8, 131.3, 130.6, 130.0, 128.6, 127.4, 127.3, 122.0, 118.4, 101.8, 99.8, 77.6, 77.2, 71.7, 55.8, 50.3, 50.0. HRMS + (ESI) m/z calc’d for C23H23Cl2N2O3 [M+H] : 445.1086. found: 445.1043.

trans-4-(2-(6-methoxyquinolin-4-yl)ethyl)cyclohexanamine (90). To a solution of compound 89 (1.05 g, 2.73 mmol) in DCM (10 mL), trifluoroacetic acid (1.5 mL) was added dropwise at room temperature. The reaction mixture was stirred at room temperature overnight and then quenched by saturated aqueous Na2CO3 solution (50 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3x30 mL). The organic layers were combined and concentrated to afford the title compound as yellow oil (822 mg) that was used without further purification.

trans-N-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-4-(2-(6-methoxyquinolin-4- yl)ethyl)cyclohexanamine (91). The title compound was prepared in 28% yield following the general reductive amination procedure and obtained as a yellow solid. 1H NMR (300 MHz, MeOD) δ: 8.54 (d, J = 4.6, 1H); 7.91 (d, J = 9.2, 1H); 7.40 (dd, J = 2.7, 9.1, 1H); 7.33 (d, J = 9.0, 1H) ; 7.33 (d, J = 1.3, 1H); 6.84 (s, 1H); 6.78 (d, J = 1.0, 2H); 4.21 (s, 4H); 3.95 (s, 3H); 3.69 (s, 2H); 3.13-3.04 (m, 2H); 2.57-2.44 (m, 1H); 2.07-1.89 (m, 4H); 1.70- 1.58 (m, 2H); 1.48-1.31 (m, 1H), 1.31-0.96 (m, 4H). 13C NMR (75 MHz, MeOD) δ: 159.5,

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150.3, 148.2, 145.0, 144.7, 144.4, 133.2, 131.2, 129.9, 123.1, 122.6, 122.3, 118.4, 118.2, 102.9, 65.6, 57.0, 56.1, 50.7, 38.7, 38.2, 33.1, 32.7, 30.7. HRMS (ESI) m/z calc’d for + C27H33N2O3 [M+H] : 433.2491; Found: 433.2471.

7-(((trans-4-(2-(6-methoxyquinolin-4-yl)ethyl)cyclohexyl)amino)methyl)-2H- pyrido[3,2-b][1,4]oxazin-3(4H)-one (93). The title compound was prepared in 34% yield following the general reductive amination procedure and obtained as a white solid. 1H

NMR (300 MHz, CDCl3) δ: 8.66 (d, J = 4.4, 1H); 8.02 (d, J = 9.2, 1H); 7.35 (dd, J = 2.7, 9.2, 1H); 7.23 (d, J = 2.7, 1H); 7.20 (d, J = 8.1, 1H); 7.17 (d, J = 4.7, 1H); 6.93 (d, J = 8.1, 1H); 4.63 (s, 2H); 3.94 (s, 3H); 3.86 (s, 2H); 3.04-2.98 (m, 2H); 2.55-2.45 (m, 1H); 2.04- 1.91 (m, 4H) ; 1.71-1.59 (m, 2H); 1.48-1.33 (m, 1H); 1.30-1.17 (m, 2H); 1.12-0.98 (m, 13 2H). C NMR (75 MHz, CDCl3) δ: 165.7, 157.7, 150.8, 147.9, 147.3, 144.5, 140.6, 138.4, 131.8, 128.5, 124.2, 121.2, 120.9, 118.4, 101.9, 67.4, 56.8, 55.7, 50.7, 37.4, 36.9, 32.7, + 31.8, 29.9. HRMS (ESI) m/z calc’d for C26H31N4O3 [M+H] : 447.2396; Found: 447.2376.

1-(2-(6-methoxyquinolin-4-yl)ethyl)piperidin-4-amine (99). To a solution of intermediate 98 (1.09 g, 2.83 mmol) in DCM (20 mL), trifluoroacetic acid (3 mL) was added dropwise at room temperature. The reaction mixture was stirred at room temperature overnight and then quenched by saturated aqueous Na2CO3 solution (100 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3x30 mL). The organic layers were combined and concentrated to afford the title compound as yellow oil (810 mg) that was used without further purification. 114

N-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-1-(2-(6-methoxyquinolin-4- yl)ethyl)piperidin-4-amine (100). The title compound was prepared in 22% yield following the general reductive amination procedure and obtained as a yellow solid. 1H NMR (300 MHz, MeOD) δ: 8.57 (d, J = 4.5, 1H); 7.95-7.92 (m, 1H); 7.46-7.40 (m, 2H); 7.38 (d, J = 4.6, 1H); 6.99 (d, J = 1.8, 1H); 6.95-6.84 (m, 2H); 4.25 (s, 4H); 4.06 (s, 2H); 3.98 (s, 3H); 3.41-3.34 (m, 4H); 3.15-3.01 (m, 1H); 2.85-2.80 (m, 2H); 2.34-2.10 (m, 4H); 1.77-1.63 (m, 2H). 13C NMR (75 MHz, MeOD) δ: 159.7, 148.2, 147.2, 146.0, 145.4, 144.8, 131.4, 130.0, 126.6, 123.7, 123.3, 123.0, 119.6, 118.8, 102.9, 65.70, 65.64, 58.6, 56.2, 55.9, 52.6, 30.5, 29.9. One peak appears to be obscured by the solvent peak. HRMS (ESI) m/z + calc’d for C26H32N3O3 [M+H] : 434.2444; Found: 434.2427.

6-(((1-(2-(6-methoxyquinolin-4-yl)ethyl)piperidin-4-yl)amino)methyl)-2H- pyrido[3,2-b][1,4]oxazin-3(4H)-one (102). The title compound was prepared in 28% yield following general reductive amination procedure and obtained as a yellow solid. 1H

NMR (300 MHz, CDCl3) δ: 8.66 (d, J = 4.4, 1H); 8.02 (d, J = 9.2, 1H); 7.36 (dd, J = 2.7, 9.2, 1H); 7.30 (d, J = 2.1, 1H); 7.23-7.17 (m, 2H); 6.94 (d, J = 8.1, 1H); 4.63 (s, 2H); 3.95 (s, 3H); 3.84 (s, 2H); 3.32-3.20 (m, 2H); 3.12-3.02 (m, 2H); 2.85-2.73 (m, 2H); 2.66-2.54 (m, 1H); 2.24 (br s, 2H); 2.00 (d, J = 11.3, 2H); 1.69-1.47 (m, 2H). 13C NMR (75 MHz,

CDCl3) δ: 165.8, 157.9, 152.1, 147.7, 145.0, 144.4, 140.6, 138.2, 131.8, 128.6, 124.1, 121.50, 121.46, 117.9, 101.8, 67.3, 58.4, 55.7, 54.5, 52.5, 51.1, 32.6, 30.1. HRMS (ESI) + m/z calc’d for C25H30N5O3 [M+H] : 448.2349; Found: 448.2329.

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2.5.2 Biological evaluation assays

Determination of Minimum Inhibitory Concentrations (MICs). MICs against the strains listed in Tables 2.1, 2.2 and 2.3 were determined by broth microdilution according to the guidelines established by the Clinical and Laboratory Standards Institute [34].

Topoisomerase Inhibition. The assays were conducted at Inspiralis, Ltd. (Norwich, UK) unless otherwise noted. In all experiments, the activity of the enzymes was determined prior to the testing of the compounds and 1 unit (U) was defined as the amount of enzyme required to just fully supercoil or decatenate the substrate. This amount of enzyme was initially used in determination of control inhibitor activity. These experiments were performed in duplicate.

For all assays the final DMSO concentration was 1%. 10 mM stocks of the compounds were serially diluted into 10% DMSO at 1 mM, 0.5 mM, 0.25 mM, 0.1mM, 0.05 mM, 0.01 mM, 0.005 mM,0.001 mM, 0.0005 mM, 0.0001 mM and 0.00005 mM. 3.0 μl of each dilution was added to a 30 μl assay. Bands were visualised by ethidium staining for 20 minutes and destaining for 20 minutes. Gels were scanned using documentation equipment (GeneGenius, Syngene, Cambridge, UK) and % inhibition levels (where appropriate) were obtained with gel scanning software. (GeneTools, Syngene, Cambridge,UK)

Staphylococcus aureus gyrase supercoiling assay. 1 U of DNA gyrase was incubated with 0.5 μg of relaxed pBR322 DNA in a 30 μl reaction at 37C for 30 minutes under the following conditions: 40 mM HEPES. KOH (pH 7.6), 10 mM magnesium acetate, 10 mM DTT, 2 mM ATP, 500 mM potassium glutamate and 0.05 mg/ml BSA. Each reaction was stopped by the addition of 30 μl chloroform/iso-amyl alcohol (26:1) and 30 μl Stop Dye (40% sucrose (w/v), 100 mM Tris.HCl (pH 7.5), 10 mM EDTA, 0.5 μg/ml bromophenol blue), before being loaded on a 1.0% TAE gel run at 70V for 2 hours.

Staphylococcus aureus topoisomerase IV decatenation assay. Decatenation assays were performed as per protocol by Inspiralis, Ltd. (Norwich, UK) where indicated (Method A).

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Briefly, per Inspiralis, decatenation of 200 ng kDNA was performed at 37C for 30 min in a total reaction volume of 30 µL containing 50 mM Tris.HCl (7.5), 5 mM MgCl2, 5 mM DTT, 1.5 mM ATP, 350 mM potassium glutamate, 0.05 mg/ml BSA, 1% DMSO vehicle control (or compound solution) and 1 unit of topoisomerase IV (defined as that amount of enzyme to just completely decatenate kDNA). Each reaction was stopped by the addition of 30 μL chloroform/isoamyl alcohol (26:1) and 30 μL of a buffer containing 40% (w/v) sucrose, 100 mM Tris-HCl (pH 8), 10 mM EDTA, 0.5 mg/mL bromophenol blue. Aqueous fractions (20 µL) were loaded onto a 1% Agarose gel and run at 70V for 2 hr, stained with ethidium bromide for subsequent UV visualization and quantitation of the percent decatenation in the presence or absence of various concentrations of test compounds, and assessment of 50% inhibitory concentrations. Where noted, results were obtained at The Ohio State University based on slight modifications (Method B) where reactions contained 100 ng kDNA, were incubated for 20 min, and contained 1.7% DMSO control (or test compound solutions). hERG Inhibition. Assays were conducted at Charles River (Cleveland, OH) using IonWorksTM Barracuda systems (Molecular Devices Corporation, Union City, CA). Evaluations were conducted using four replicates per concentration for each compound. Cisapride was employed as the positive control.

Growth Inhibition. Log-phase parental K562 and cloned K/VP.5 cells were adjusted to 1 x 105 cell/ml and 1.25 x 105 cell/mL respectively, and incubated for 48 hr with 0-200 μM NBTIs after which cells were counted on a model ZBF Coulter counter (Beckman Coulter, Danvers, MA). Growth beyond the starting concentrations in drug-treated versus control cells was ultimately expressed as percent inhibition of control growth. The 50% growth-inhibitory concentration for each NBTI in each cell line was calculated from concentration-response curves generated by use of Sigmaplot 13 (Systat Software, Inc; San Jose, CA). All NBTI were dissolved in 100% DMSO and added to cell suspensions to achieve a final solvent concentration of 0.5%.

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2.6 Reference

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region in the DNA gyrase gyrA gene of Escherichia coli. Antimicrob Agents Chemother 1990, 34, 1271-1272. 16. Singh SB, Kaelin DE, Wu J, Miesel L, Tan CM, Meinke PT, et al. Oxabicyclooctane- Linked Novel Bacterial Topoisomerase Inhibitors as Broad Spectrum Antibacterial Agents. ACS Med Chem Lett 2014, 5, 609-614. 17. Singh SB, Kaelin DE, Wu J, Miesel L, Tan CM, Black T, et al. Tricyclic 1,5- naphthyridinone oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents-SAR of left-hand-side moiety (Part-2). Bioorg Med Chem Lett 2015, 25, 1831-1835. 18. Tan CM, Gill CJ, Wu J, Toussaint N, Yin J, Tsuchiya T, et al. In Vitro and In Vivo Characterization of the Novel Oxabicyclooctane-Linked Bacterial Topoisomerase Inhibitor AM-8722, a Selective, Potent Inhibitor of Bacterial DNA Gyrase. Antimicrob Agents Chemother 2016, 60, 4830. 19. Surivet JP, Zumbrunn C, Rueedi G, Hubschwerlen C, Bur D, Bruyere T, et al. Design, synthesis, and characterization of novel tetrahydropyran-based bacterial topoisomerase inhibitors with potent anti-gram-positive activity. J Med Chem 2013, 56, 7396-7415. 20. Surivet JP, Zumbrunn C, Rueedi G, Bur D, Bruyere T, Locher H, Ritz D, et al. Novel tetrahydropyran-based bacterial topoisomerase inhibitors with potent anti-gram positive activity and improved safety profile. J Med Chem 2015, 58, 927-942. 21. Surivet JP, Zumbrunn C, Bruyére T, Bur D, Kohl C, Locher HH, Seiler P, et al. Synthesis and Characterization of Tetrahydropyran-Based Bacterial Topoisomerase Inhibitors with Antibacterial Activity against Gram-Negative Bacteria. J Med Chem 2017, 60, 3776-3794. 22. Dougherty TJ, Nayar A, Newman JV, Hopkins S, et al. NBTI 5463 is a novel bacterial type II topoisomerase inhibitor with activity against gram-negative bacteria and in vivo efficacy. Antimicrob Agents Chemother 2014, 58, 2657-2664. 23. Miles TJ, Hennessy AJ, Bax B, Brooks G, Brown BS, et al. Novel hydroxyl tricyclics (e.g., GSK966587) as potent inhibitors of bacterial type IIA topoisomerases. Bioorg Med Chem Lett 2013, 23, 5437-5441. 24. Kriek NMA, van der Hout E, Kelly P, van Meijgaarden KE, Geluk A, et al. Synthesis of Novel Tetrahydropyran-Based Dipeptide Isosteres by Overman Rearrangement of 2,3-Didehydroglycosides. Eur J Org Chem 2003, 2418-2427. 25. Reck F, Alm R, Brassil P, Newman J, et al. Novel N-linked aminopiperidine inhibitors of bacterial topoisomerase type II: broad-spectrum antibacterial agents with reduced hERG activity. J Med Chem 2011, 54, 7834-7847. 26. Reck F, Alm RA, Brassil P, Newman JV, Ciaccio P, et al. Novel N-Linked Aminopiperidine Inhibitors of Bacterial Topoisomerase Type II with Reduced pKa: Antibacterial Agents with an Improved Safety Profile. J Med Chem 2012, 55, 6916- 6933. 27. Ndubaku CO, Crawford JJ, Drobnick J, Aliagas I, Campbell D, Dong P, et al. Design of Selective PAK1 Inhibitor G-5555: Improving Properties by Employing an Unorthodox Low-pKa Polar Moiety. ACS Med Chem Lett 2015, 6, 1241-1246. 28. Lall MS, Tao Y, Arcari JT, Boyles D, et al. Process Development for the Synthesis of

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Monocyclic β-Lactam Core 17. Org Process Res Dev 2018, 22, 212-218. 29. Li L, Okumu A, Dellos-Nolan S, et al. 1,3-Dioxane-linked Bacterial Topoisomerase Inhibitors with Enhanced Antibacterial Activity and Reduced hERG Inhibition. ACS Infect Dis 2019, 5, 1115-1128. 30. Barfoot CW, Brown P, Dabbs S, Davies DT, et al. The design of efficient and selective routes to pyridyl analogues of 2,3-dihydro-1,4-benzodioxin-6-carbaldehyde. Tetrahedron Lett 2010, 51, 5038-5040. 31. Sonogashira K. Development of Pd–Cu catalyzed cross-coupling of terminal acetylenes with sp2-carbon halides. J Organomet Chem 2002, 653, 46-49. 32. Miller WH, Price AT. Antibacterial agents. WO 2007118130 A2, Oct. 18, 2007. 33. Chen X, Zhang Y, Wan H, Wang W, Zhang S. Stereoselective organocatalytic oxidation of alcohols to enals: a homologation method to prepare polyenes. Chem Commun 2016, 52, 3532-3535. 34. Clinical and Laboratory Standards Institute (CLSI). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard—Eleventh Edition. CLSI document M07-Ed11. Clinical and Laboratory Standards Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087 USA, 2018. 35. Zafrani Y, Sod-Moriah G, Yeffet D, Berliner A, et al. CF2H, a Functional Group- Dependent Hydrogen-Bond Donor: Is It a More or Less Lipophilic Bioisostere of OH, SH, and CH3? J Med Chem 2019, 62, 5628-5637. 36. Hidron AI, Low CE, Honig EG, Blumberg HM. Emergence of community-acquired meticillin-resistant Staphylococcus aureus strain USA300 as a cause of necrotising community-onset pneumonia. Lancet Infect Dis 2009, 9, 384-392. 37. Wang JC. DNA topoisomerases. Annu Rev Biochem 1996, 65, 635-692. 38. Ashley RE, Lindsey RH, McPherson SA, Turnbough CL, et al. Interactions between Quinolones and Bacillus anthracis Gyrase and the Basis of Drug Resistance. Biochemistry 2017, 56, 4191-4200. 39. Varughese LR, Rajpoot M, Goyal S, Mehra R, et al. Analytical profiling of mutations in quinolone resistance determining region of gyrA gene among UPEC. PLoS One 2018, 13, e0190729. 40. Kanagasabai R, Serdar L, Karmahapatra S, Kientz CA, et al. Alternative RNA Processing of Topoisomerase IIα in Etoposide-Resistant Human Leukemia K562 Cells: Intron Retention Results in a Novel C-Terminal Truncated 90-kDa Isoform. J Pharm Exptl Ther 2017, 360, 152-163. 41. The observed splitting pattern is characteristic of the trans-substituted dioxane and distinct from the cis isomer. For earlier work, see: Bailey WF, Lambert KM, Stempel ZD, Wiberg KB, Mercado BO. Controlling the Conformational Energy of a Phenyl Group by Tuning the Strength of a Nonclassical CH···O Hydrogen Bond: The Case of 5-Phenyl-1,3-dioxane. J Org Chem 2016, 81, 12116-12127. 42. a) Binev Y, Marques MMB, Aires-de-Sousa J. Prediction of 1H NMR Coupling Constants with Associative Neural Networks Trained for Chemical Shifts. J Chem Inf Model 2007, 47, 2089. b) Online simulation conducted at www.nmrdb.org.

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Chapter 3. Optimization of 5-amino-1,3-dioxane

Linked NBTIs

3.1 Abstract

In Chapter 2, the use of a 5-amino-1,3-dioxane linker moiety in NBTIs was concluded to reduce hERG inhibition, and a number of dioxane-linked NBTIs possessed good anti- staphylococcal activity. In this chapter, several new NBTIs with the 5-amino-1,3-dioxane linker were developed and synthesized. Most of these new NBTIs display efficacious whole cell activity and target both DNA gyrase and topoisomerase IV potently. Nevertheless, inhibition of topoisomerase IV still needs to be optimized. Favorable hERG profiles were obtained for several new NBTIs. In preliminary in vitro human safety experiments, these new NBTIs were shown to have low toxicity to human cells and to spare inhibition of the homologous human topoisomerase IIα.

3.2 Introduction

As described in previous chapters, noteworthy progress in the discovery of new NBTIs has been documented recently [1, 2]. The safety issue of hERG inhibition, which can cause cardiac danger, has been commonly reported in NBTI projects. [3, 4] In Chapter 2, the 5- amino-1,3-dioxane moiety was demonstrated to reduce hERG inhibition of NBTIs, and some dioxane-linked NBTIs using bicyclic moieties as the enzyme-binding domain were developed with promising biochemical properties. Based on these studies, further optimization of the DNA-binding domain in new NBTIs with 5-amino-1,3-dioxane linker was pursued in this chapter, aiming to obtain remarkable NBTIs with promising potency and a favorable hERG profile.

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Figure 3.1 AstraZeneca NBTIs with N-linked quinoxalinone moiety: A [4], B [5]

Recently, the N-linked quinoxalinone was developed as a DNA-binding moiety in NBTIs discovered by AstraZeneca (Fig 3.1, AstraZeneca-24 [5]). Compared with the previously reported AstraZeneca-2 (Fig 3.1) with a C-linked fluoronaphthyridine DNA- binding moiety, AstraZeneca-24 demonstrated an improved hERG profile (hERG IC50=2 M and 19 M, respectively) and maintained the in vitro broad spectrum antibacterial activity and in vivo efficacy against S. aureus in a neutropenic thigh infection model [5]. The 7-fluoro-2-methoxy-1,5-naphthyridine of AstraZeneca-2 has been widely used as a DNA-binding moiety in other reported NBTIs as well [6-8]. For example, AM8085 and AM8191 (figure 3.2) disclosed by Merck [6] showed good whole cell activity against Gram-positive and Gram-negative strains, including MRSA and quinolone-resistant strains.

The hERG inhibition of AM8085 (IC50=0.6 μM) was very potent but could be reduced significantly by the addition of a hydroxyl group at C-2 in the linker (AM8191, IC50=18 μM). Moreover, the antibacterial potency of AM8191 was also demonstrated in vivo in murine models of S. aureus and E. coli infections [6]. Utilizing the 7-fluoro-2-methoxy- 1,5-naphthyridine as the DNA-binding moiety, Actelion-5 and ACT-387042 (Fig 3.30) also displayed strong Gram-positive potency [7]. The in vivo efficacy of Actelion-5 against S. pneumoniae and S. aureus was confirmed in a neutropenic murine thigh infection model [8]. Although the hERG block of the diol-containing Actelion-5 (19% block at 10 μM) was not particularly intense, it was found to affect various cardiovascular parameters in anesthetized guinea pigs [7]. With the mono-hydroxylated linker and use of the more polar oxathiinopyridazine enzyme-binding moiety, ACT-387042 reduced the inhibition of the hERG K+ channel (10% block at 10 μM) and showed no effects on cardiovascular safety parameters in the guinea pig at free concentrations up to 8 μM. Meanwhile, ACT-387042 retained promising Gram-positive potency, a low frequency of resistance, and good in vivo efficacy, which resulted in the initiation of a preclinical development program for ACT- 122

387042 [7-9].

O H HO O H N N O N N O N N H H O N F O O N F O

N AM8085 N AM8191

HO O HO O S S HO N N H H N N O N F O O N F N O

N ACT-387042 N Actelion-5 Figure 3.2 Merck NBTIs [6]: AM8085 and AM8191). Actelion NBTIs [7]: Actelion-5 and ACT-387042.

Taking advantage of those moieties with good preclinical properties from previously reported NBTIs and considering synthetic convenience, several components were employed as DNA-binding moieties in our new NBTIs. In this work, the polar N-linked quinoxalinone moiety was embodied into dioxane-linked NBTIs (Figure 3.3, A), with the purpose of maintaining antibacterial activity and improving the hERG profile over NBTIs with the 6-methoxyquinoline DNA-binding moiety from Chapter 2. 7-fluoro-2-methoxy- 1,5-naphthyridine analogs with the dioxane linker were also developed in this chapter (Figure 3.3, B). Anticipating similar promising preclinical properties to AM-8191 and ACT-387042, several analogs with a hydroxyl group at either the C-1 or C-2 position of the linker were incorporated in our new NBTIs, including 1-(3-fluoro-6-methoxy -1,5- naphthyridin-4-yl)ethan-1-ol analogs (Figure 3.3, C), 1-(3-fluoro-6- methoxyquinolin-4-yl) ethan-1-ol analogs (Figure 3.3, D), and 2-(7-fluoro-2- methoxyquinolin-8-yl)ethan-1-ol analogs (Figure 3.3, E). Based on the SAR study on the enzyme-binding moieties of 1,3- dioxane linked NBTIs reported in Chapter 2, promising bicyclic enzyme-binding moieties were primarily utilized in the new NBTIs.

123

Figure 3.3 Structures of new 5-amino-1,3-dioxane linked NBTIs

Whole cell anti-staphylococcal activity, as well as DNA gyrase and topoisomerase IV target inhibition for these new NBTIs were tested in vitro, and promising compounds were selected for further study. Since the binding mode of NBTIs is distinct from that of fluoroquinolones [1], no cross resistance is expected between fluoroquinolones and our new compounds. Hence, selected compounds were assayed to evaluate the activity against gyrase with fluoroquinolone-resistant mutations. Broad-spectrum antibacterial activity and spontaneous frequency of resistance were also tested for representative compounds. As the key concern of this work, hERG inhibition was assessed for all potent new NBTIs. In order to further address human safety issues, selectivity over the homologous human topoisomerase IIα and toxicity in a human leukemia cell line were also tested in vitro for these newly synthesized NBTIs.

3.3 Results and Discussion

3.3.1 Synthesis

124

3.3.1.1 Synthesis of N-linked quinoxalinone analogs

Scheme 3.1 Synthesis of N-linked quinoxalinone analogs. Reaction conditions: i. Dess-Martin periodinane, DCM, 90%; ii. p-TsOH (cat.), toluene, 110 °C, 67%; iii. Pd/C, H2, MeOH, 89%; iv. methanesulfonic anhydride, pyridine, 81%; v. ethyl bromoacetate, K2CO3, 150 °C, 79%; vi. H2, Pd/C, MeOH/AcOH, used directly; vii. a) 30 wt% H2O2, H2O, 80 °C. b) AcOH, 51% for two steps; viii. compound 6, Cs2CO3, DMSO, 36%; ix. HO(CH2)2NH2, EtOAc, reflux, 76%; x. NaBH3CN, ZnCl2, MeOH.

Compounds with a quinoxalinone DNA-binding moiety were reported to have good broad-spectrum antibacterial activity in previous NBTIs [10]. Four compounds incorporating an N-linked quinoxalinone moiety and 5-amino-1,3-dioxane linker 125

(compounds 13-16) were synthesized. The synthetic route is presented in Scheme 3.1. Dess-Martin oxidation of the commercially available 3-(benzyloxy)propan-1-ol 1 afforded 3-(benzyloxy)propanal 2. Condensation of 3-(benzyloxy)propanal 2 and diol 3 (Intermediate 5 in Chapter 2, Scheme 2.1) resulted in 1,3-dioxane 4. Deprotection of the benzyl group by Pd-catalyzed hydrogenolysis gave rise to alcohol 5, which was transformed into mesylate 6. The alkylation of commercially available 4-methoxy-2- nitrobenzenamine 7 with ethyl bromoacetate led to compound 8. Reduction of the nitro group and cascade cyclization yielded bicyclic compound 9, which was directly oxidized into 7-methoxy-quinoxalinone 10. Alkylation of quinoxalinone 10 with mesylate 6 produced N-linked quinoxalinone 11 successfully. The N- or O-alkylation was determined 1 by a 1D selective gradient H NOESY experiment. In the NOESY experiment, H1 and H2 in compound 11 showed no correlation with each other, indicating that the two protons are not proximity in space and consistent with the N-analogue. Deprotection of the phthalimide yielded the primary amine 12. Finally, reductive amination with the relevant aldehydes afforded target compounds 13-16.

126

3.3.1.2 Synthesis of 7-fluoro-2-methoxy-1,5-naphthyridine analogs

Scheme 3.2 Synthesis of 7-fluoro-2-methoxy-1,5-naphthyridine analogs. Reaction conditions: i. acrolein, CuSO4, p-TsOH (cat.), DCM, 76%; ii. DMF, PBr3, 90%; iii. a) 9-BBN, THF, 30 min. b) Naphthyridine bromide 19, Pd(PPh3)2Cl2, Cs2CO3, THF, 58%; iv. HO(CH2)2NH2, EtOAc, reflux, 80%; v. NaBH3CN, ZnCl2, MeOH.

As introduced before, 7-fluoro-2-methoxy-1,5-naphthyridine has been widely used as a DNA-binding moiety in previously documented NBTIs [6, 7]. Herein, seven compounds with this naphthyridine DNA-binding moiety were synthesized. The route shown in Scheme 3.2 is based on a slight modification of the synthesis of 6-methoxyquinoline analogs from Chapter 2. Catalyzed by p-toluenesulfonic acid, condensation of diol 3 with acrolein proceeded to alkene 17 (intermediate 6 in chapter 2, scheme 2.1). Naphthyridine bromide 19 was obtained in high purity (>95%) via bromination of the commercially available 3-fluoro-6-methoxy-1,5-naphthyridin-4-ol 18 with PBr3. A Suzuki Coupling between bromide 19 and the hydroborated intermediate derived from alkene 17 afforded compound 20, which was then deprotected to provide primary amine 21. Final analogs 22-

127

29 were synthesized via reductive amination of the primary amine 21 with the requisite aldehydes under our standard conditions.

3.3.1.3 Synthesis of 1-(3-fluoro-6-methoxy-1,5-naphthyridin-4-yl)ethan-1-ol analogs

Scheme 3.3 Synthesis of 1-(3-fluoro-6-methoxy-1,5-naphthyridin-4-yl)ethan-1-ol analogs. Reaction conditions: i. p-TsOH (cat.), toluene, 85 °C, 26%; ii. pyridinium p-toluenesulfonate, THF/H2O, 80 °C, 31%; iii. a) n-hexyllithium, THF, -78 °C. b) aldehyde 32, THF, -78 °C, 18%; iv. HO(CH2)2NH2, EtOAc, reflux, 66%; v. NaBH3CN, ZnCl2, MeOH.

In previous NBTIs like Actelion-5 (Figure 3.2) [7], the addition of a hydroxy group in the linker part afforded an improved hERG profile. Herein, six compounds with a 1-(3- fluoro-6-methoxy-1,5-naphthyridin-4-yl)ethan-1-ol moiety were synthesized. The pathway is depicted in Scheme 3.3. The condensation of diol 3 with commercially available 1,1,3,3-tetramethoxypropane 30 led to dimethoxy acetal 31 under catalysis by p- toluenesulfonic acid. Selective acid-catalyzed hydrolysis of the acyclic dimethyl acetal 31 128

[11] afforded aldehyde 32. Lithium-halogen exchange of naphthyridine bromide 19 and n- hexyllithium afforded the lithiated intermediate, which was then reacted with aldehyde 32 to give rise to alcohol 33. The newly formed chiral center in this step was racemic, and the resulting analogs were also prepared as racemates. Unfortunately, this step proceeded with a very low yield due to competitive reaction with the carbonyl group of the phthalimide protecting group. Deprotection of the phthalimide resulted in the primary amine 34. Final analogs (compounds 35-40) were synthesized via reductive amination of the primary amine 34 with the relevant aldehydes. These racemic final compounds were evaluated in subsequent biological tests without chiral separation.

3.3.1.4 Synthesis of 2-(5-(((6,7-dihydro-[1,4]oxathiino[2,3-c]pyridazin-3-yl) methyl)amino)-trans-1,3-dioxan-2-yl)-1-(3-fluoro-6-methoxyquinolin-4-yl)ethan-1-ol

Scheme 3.4 Synthesis of 2-(5-(((6,7-dihydro-[1,4]oxathiino[2,3-c]pyridazin-3-yl) methyl)amino)-trans-1,3- dioxan-2-yl)-1-(3-fluoro-6-methoxyquinolin-4-yl)ethan-1-ol. Reaction conditions: i. a) quinoline 41, LDA, THF, -78 °C. b) aldehyde 32, THF, -78 °C, 10%; ii. HO(CH2)2NH2, EtOAc, reflux, used directly; iii. Aldehyde, NaBH3CN, ZnCl2, MeOH, 51%.

Additional NBTIs with a hydroxylated linker were pursued due to the potential to improve the hERG profile and to reduce metabolism while maintaining potent whole cell activity [6, 7]. Dr. Antony Okumu in our lab had been pursuing NBTIs analogs with 3- fluoro-6-methoxyquinolin-4-yl)ethan-1-ol moiety. Among those compounds, 2-(5-(((6,7- dihydro-[1,4]oxathiino[2,3-c]pyridazin-3-yl)methyl)amino)-trans-1,3-dioxan-2-yl)-1-(3- fluoro-6-methoxyquinolin-4-yl)ethan-1-ol displayed good preliminary potency. It was resynthesized by a similar route to the fluoronaphthyridine analogs (Scheme 3.4) in this

129 work to support additional studies. Deprotonation of the commercially available fluoroquinoline 41 with lithium diisopropylamide produced fresh lithium-quinoline intermediate, which was directly reacted with aldehyde 32 to give rise to alcohol 42. The low yield of this step again arose from competitive reaction with the carbonyl group of the phthalimide moiety. Deprotection of phthalimide of compound 42 resulted in the primary amine 43. Reductive amination of primary amine 43 with 6,7-dihydro-[1,4]oxathiino[2,3- c]pyridazine-3-carbaldehyde afforded final compound 44.

130

3.3.1.5 Synthesis of 2-(7-fluoro-2-methoxyquinolin-8-yl)ethan-1-ol analogs

Scheme 3.5 Synthesis of 2-(7-fluoro-2-methoxyquinolin-8-yl)ethan-1-ol analogs. Reaction conditions: i. a) sodium bis(trimethylsilyl)amide, THF, 0 °C-25 °C. b) H2SO4 (conc.), DCM, 0 °C; ii. POCl3, DMF, toluene, 95 °C; iii. NaOMe, toluene, 80 °C; iv. Ag2CO3, Pd(OAc)2 (cat.), PPh3 (cat.), mesitylene, 135 °C; v. HO(CH2)2NH2, EtOAc, reflux; vi. di-tert-butyl dicarbonate, triethylamine, DCM; vii. AD mix β, methanesulfonamide, ethyl acetate/H2O/tert-butanol; viii. 1,1'-carbonyldiimidazole, triethylamine, 2- butanone, 60 °C; ix. ammonium formate, 5% palladium on calcium carbonate, 10% palladium on carbon, ethyl acetate/ethanol, 70 °C-40 °C; x. trifluoroacetic acid, DCM; xi. NaBH3CN, ZnCl2, MeOH. 131

NBTIs with a hydroxyl group in the C-2 position of the linker have been shown to reduce hERG inhibition, such as AM8191 [6] and ACT-387042 [7]. In this project, new NBTIs with a C-2 hydroxylated linker, 2-(7-fluoro-2-methoxyquinolin-8-yl)ethan-1-ol analogs (57-61), were synthesized, as presented in scheme 3.5. The pathway to 2- methoxyquinoline 49 was similar to that reported for other 2-methoxyquinoline derivatives [12]. Acylation of commercially available 2-bromo-3-fluoroaniline 45 with commercially available methyl 3,3-dimethoxypropanoate 46 was followed by a condensation promoted by sulfuric acid, resulting in quinolinone 47. Then quinolinone 47 was converted to 2- chloroquinoline 48 via the addition of POCl3. The 2-chloroquinoline 48 was transformed to 2-methoxyquinoline 49 by reaction with sodium methoxide [12]. A Heck coupling between 2-methoxyquinoline 49 and alkene 17 gave rise to compound 50. In this step, m- xylene was originally used as solvent, but m-xylene was observed to undergo coupling with the alkene 17, resulting in xylene derivatives as side products which were difficult to separate. Mesitylene was then used as an alternative solvent and avoided similar side reactions due to additional steric hindrance of reactive sites on the phenyl ring. We sought to transform the alkene group in compound 50 into a dihydroxyl moiety, but the poor solubility of compound 50 (due to the phthalimide) precluded efficient dihydroxylation. Hence, the phthalimide protecting group in compound 50 was transferred into a tert- butyloxycarbonyl protecting group in compound 52 through a deprotection/reprotection sequence. Then the alkene group in compound 52 underwent asymmetric dihydroxylation under the conditions of Sharpless using AD mix β [14] to afford diol 53, analogously to the dihydroxylation leading to Actelion-5 [13]. The enantiomeric excess value of diol 53 was not determined, but enantio-selectivity of the asymmetric dihydroxylation was presumed to be higher than 95% based on literature [13, 14]. The cyclic carbonate 54 was then constructed by reaction of the diol with 1,1'-carbonyldiimidazole. Reduction of the cyclic carbonate group released the (S)-configured hydroxyl in alcohol 55, again mimicking the synthetic process leading to ACT-387042 [7]. Acidic deprotection of the tert-butyloxycarbonyl group with trifluoroacetic acid resulted in the primary amine 56. Final analogs (compounds 57-61) were synthesized via reductive amination of primary amine 56 with the requisite aldehydes under our standard conditions. 132

3.3.1.6 Efforts to optimize the synthesis of NBTIs with C-2 hydroxyl linker

Scheme 3.6 Proposed alternative synthetic pathway to 2-(5-amino-trans-1,3-dioxan-2-yl)-1-(7-fluoro-2- methoxyquinolin-8-yl)ethan-1-ol primary amine. Reaction conditions: i. Diisobutylaluminium hydride, ether, -78 °C, N2, used directly; ii. a) n-hexyllithium, THF, -78 °C. b) aldehyde 63, THF, -78 °C, 74% over two steps; iii. BF3·O(C2H5)2, chloroform, reflux, 0%; iv. HO(CH2)2NH2, EtOAc, reflux; v. BF3·O(C2H5)2, chloroform, reflux, 62%.

Both the synthesis of fluoronaphthyridine analogs (35-40) and the synthesis of the fluoroquinoline analog (44) suffered from very low overall yields, mainly due to preferential reaction with the carbonyl group of the phthalimide protecting group rather than the aldehyde moiety. In order to optimize this step, we sought an approach in which the dioxane would be installed after the coupling reaction. This strategy would also be 133 amenable to the synthesis of analogs with diverse DNA-binding moieties. One approach we pursued is shown in scheme 3.6, with the goal of synthesizing primary amine 66. The commercially available ethyl 3,3-diethoxypropionate 62 was reduced into aldehyde 63 with diisobutylaluminium hydride. Metal-halogen exchange of quinoline bromide 49 and n-hexyllithium afforded the lithiated intermediate, which was then reacted in situ with aldehyde 63 to produce acetal alcohol 64. Condensation of diol 3 with the acetal of compound 64 was endeavored in order to produce the 1,3-dioxane 65 under acidic catalysis. Through deprotection of the phthalimide moiety, the 1,3-dioxane 65 would be transformed to the target primary amine 66. However, no desired 1,3-dioxane 65 was observed. Instead of the envisioned intermolecular condensation, the acetal alcohol 64 underwent a Lewis acid-catalyzed rearrangement, resulting in tricyclic compound 67 as the major product in 62% yield. A proposed mechanism of this rearrangement is depicted in scheme 3.6. Presumably, application of this approach to the synthesis of fluoronaphthyrdine analogs would suffer from the same complications, and additional optimization of this route to 1,3- dioxane analogs was not pursued. Additionally, the obtained tricyclic structure of 67 has the potential to be modified and applied in new NBTIs as innovative tricyclic DNA-binding moieties, which will be studied in future plans.

In summary, several new 5-amino-1,3-dioxane linked analogs have been synthesized. The synthesis of N-linked quinoxalinone analogs (13-16) and fluoronaphthyridine analogs (22-29) proceeded in good overall yield. Although undesired side products were obtained during coupling reactions of lithiated DNA-binding moieties with phthalimide-protected dioxane intermediates, hydroxylated fluoronaphthyridine analogs (35-40) and the hydroxylated fluoroquinoline analog 44 were nevertheless prepared successfully, albeit in low yield. Similar to the synthesis of ACT-387042 [7], the C-2 hydroxyl group was installed in the linker of fluoronaphthyridine analogs (57-61). These newly prepared NBTIs were then evaluated in a variety of biological assays.

134

3.3.2 Biological evaluation of new NBTI analogs

3.3.2.1 Minimum inhibitory concentrations

NBTIs hold promise as a new class of antibiotics. Due to the distinct mechanism of action, NBTIs are expected to possess potent activity against pre-existing multidrug- resistant organisms, especially fluoroquinolone-resistant strains [1, 2]. Several 1,3- dioxane-linked NBTIs with bicyclic moieties presented good whole cell antibacterial activity in Chapter 2. Consequently, potent anti-staphylococcal activity of the newly synthesized analogs was also expected. To evaluate whole cell potency of these new NBTIs, minimal inhibitory concentrations (MICs) for all new analogs were determined according to Clinical and Laboratory Standards Institute (CLSI) guidelines using the S. aureus reference strain ATCC 29213 and a ciprofloxacin-resistant USA 300 strain of MRSA. Ciprofloxacin was used as positive control for the ATCC 29213 strain, and vancomycin was used as the positive control for USA 300 strain. All of the MIC assays in this chapter were completed by our collaborator, Sheri Dellos-Nolan in the laboratory of Professor Wozniak at The Ohio State University [15].

As shown in Table 3.1, the four N-linked 7-methoxyquinoxalinone NBTIs in our dioxane series (13-16) possessed insufficient antibacterial activity (≥4 g/mL) against both the ATCC 29213 (untreated S. aureus strain) and the USA 300 (MRSA strain), in contrast to previous observations with piperidine-linked NBTIs. Gratifyingly, fluoronaphthyridine analogs bearing bicyclic enzyme-binding moieties (22-26, 28) displayed very potent MICs (≤0.25 g/mL) against both the ATCC and MRSA strains, as did the 3,4-dichlorophenyl derivative 27. Although the 3,4-difluorophenyl derivative 29 retained moderate MIC values (2 g/mL against ATCC29213, 4 g/mL against USA 300), it showed significantly improved activity as compared to the corresponding 6- methoxyquinoline analog (compound 46 in Chapter 2, 64 g/mL). Similarly to the naphthyridine analogs above, (1-hydroxy) naphthyridin-4-yl analogs (35-40), the (1- hydroxy) quinolin-4-yl compound 44, and (2-hydroxy) quinolin-8-yl analogs (57-61) all delivered very potent activity (≤1 g/mL) against both the ATCC and MRSA strains.

135

Moreover, the MIC data showed that most of these new analogs were as potent as the positive controls, ciprofloxacin and vancomycin, in terms of their anti-staphylococcal activity.

Of note, the USA 300 strain is a fluoroquinolone-resistant MRSA isolate, against which ciprofloxacin showed poor activity (MIC = 16-32 g/mL). Compared with ciprofloxacin, these newly synthesized analogs (22-29, 35-40, 44, 57-61) possessed much more potent USA 300 activity, supporting the value of NBTIs against fluoroquinolone- resistant bacteria. These potent compounds were also assayed against additional fluoroquinolone-resistant strains as described below.

Table 3.1．MICs for selected compounds in MSSA and MRSA cellular inhibitory experiments Compound S. aureus S. aureus Compound S. aureus S. aureus (MSSA) ATCC (MRSA) (MSSA) (MRSA) 29213 USA 300 ATCC 29213 USA 300 MIC(g/mL)a,b MIC MIC(g/mL) MIC (g/mL)a,b a,b (g/mL)a,b

13 4-32 32 36 0.5, 1 1

14 8-32 32 37 ≤0.25, 0.25 0.125, 0.25

15 8-16 32 38 0.125-0.25 0.125, ≤0.06

16 4-16 >64 39 ≤0.25, 0.25 0.25

22 ≤0.25, 0.25 0.125 40 0.5 1

23 0.125 0.125-0.25 44 1 1

24 ≤0.25, 0.125 0.25 57 0.125 0.25

25 0.125-0.5 0.25, 0.125 58 0.25 0.5

26 0.125- 2 0.25 59 0.5 0.25

27 0.25 0.125 60 0.25 0.5

28 0.25 0.25 61 0.5 0.5

136

29 2 4 ciprofloxacin 0.125-0.5 16-32

35 0.25, <=0.25 0.25 vancomycin NTc 1-2

aAssays were conducted according to CLSI guidelines. bRanges of observed values provided where appropriate. cNT = not tested

Two additional previously reported fluoroquinolone-resistant S. aureus strains were subsequently employed [31]. SA-3527 is an S. aureus strain that contains an S84L amino acid substitution in gyrase [16], one of the most frequently encountered mutations associated with quinolone resistance [18]. The 1490-09 S. aureus strain is a first-step NBTI-resistant mutant derived from SA-3527, which has an additional gyrase D83N mutation. Since the D83 residue at the entrance of the hydrophobic binding pocket of DNA gyrase can form a critical hydrogen bond (or ionic interaction) with NBTIs, amino acid substitution at this position has been frequently associated with NBTI resistance in S. aureus. A prominent early example was described in mechanism of action studies on NXL101 [18].

As shown in Table 3.2, all the new compounds inhibited the SA-3527 strain with promising MICs (most ≤0.5 g/mL), displaying excellent activity against this fluoroquinolone-resistant strain. Once again, these observations substantiate the hypothesis that NBTIs will not be impacted by fluoroquinolone resistance, as a result of their different binding mode with DNA gyrase. However, the selected NBTIs displayed much more modest MICs (2-32 g/mL) against the 1490-08 strain compared with the positive control vancomycin (MIC=1-2 g/mL). These results indicate that the gyrase D83N mutation impairs the activity of these NBTIs as expected, and demonstrate that the whole cell anti- staphylococcal activity of these newly prepared NBTIs derives mainly from the inhibition of bacterial DNA gyrase.

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Table 3.2 MICs for ciprofloxacin-resistant strain SA-3527 and 1490-08 Compound S. aureus S. aureus Compound S. aureus S. aureus (MRSA) SA- (MRSA) (MRSA) SA- (MRSA) 3527 MIC50 1490-08 3527 MIC50 1490-08 a,b a,b (g/mL) MIC50 (g/mL) MIC50 (g/mL)a,b (g/mL)a,b

22 ≤0.25 4 38 ≤0.25, ≤0.06 4

23 ≤0.25, 0.25 4 39 ≤0.25 4

24 ≤0.25 4 40 0.5 8

25 ≤0.25 8-32 44 1 >8

26 0.5-2 16-32 57 0.125 2

27 ≤0.25 4 58 0.5 4

28 0.125, ≤0.5 2-4 59 0.25 4

35 ≤0.25, 0.25 8 60 0.25 2

36 1 16 61 0.5 4

37 ≤0.25, 0.25 2 vancomycin 1-2 1-2

aAssays were conducted according to CLSI guidelines bRanges of observed values provided where appropriate.

Based on the potent anti-staphylococcal activity of these new analogs, the potential for broader spectrum activity was also evaluated. MICs for three naphthyridine analogs (22, 23, and 28) were determined against a variety of Gram-positive and Gram-negative pathogens, using ciprofloxacin as positive control. These assays were conducted at Laboratory Specialists (Westlake, OH). As shown in Table 3.3, all three compounds showed potent whole cell activity for Gram-positive pathogens, including penicillin- resistant Streptococcus pneumoniae, vancomycin-resistant Enterococcus faecium (VRE) and an additional strain of fluoroquinolone-resistant MRSA. The MIC90 value gives the

MIC which would cover 90% of all the tested strains. The MIC90 is thus a reflection of potency against different resistant strains. To our delight, compound 28 displayed great

138 potency against 11 additional strains of MRSA, despite high-level ciprofloxacin resistance

(MIC90=0.12 mg/mL and 32 mg/mL for compound 28 and ciprofloxacin, respectively). The three compounds were considerably less active against most Gram-negative organisms, which is not unexpected considering the limited drug penetration and powerful efflux pumps of Gram-negative bacteria [19, 20]. Of note, good anti-Gram-negative activity was observed for all three compounds against Acinetobacter baumannii, with compound 28 even more potent than ciprofloxacin against this strain. These results raise the hope that further optimization on these new analogs could reveal new leads for treating important Gram-negative pathogens, as has been observed with some previously documented anti- Gram-negative NBTIs [21, 22]. Further optimization of these analogs will be required to achieve this objective.

Table 3.3 Broad spectrum antibacterial activity of select NBTIs Strain Cmpd. 22 Cmpd. 23 Cmpd. 28 Ciprofloxacin

MIC MIC MIC MIC

(g/mL)a (g/mL)a (g/mL)a (g/mL)a

S. aureus ATCC 29213 (n=3) ≤0.06b ≤0.06b ≤0.06b 0.25

MRSA DRL-3161 ≤0.06 ≤0.06 ≤0.06 >64

Streptococcus pyogenes LSI-1235 ≤0.06 ≤0.06 ≤0.06 0.25

Streptococcus pneumoniae LSI-4234c 0.25 0.5 0.25 2

Streptococcus pneumoniae LSI-4246d ≤0.06 ≤0.06 ≤0.06 0.5

Enterococcus faecium DRL-4193e 0.12f 0.5 0.25g 0.25

Enterococcus faecium LSI-3340h 0.12 0.25 0.25 16

Enterococcus faecalis ATCC 29212 0.5 1 0.5 0.5

Fluoroquinolone-resistant S. aureus NT NT 0.12 32

139

(11 strains) (MIC90) (MIC90)

Escherichia coli ATCC 25922 (n=2) 16 64 32 ≤0.06

Pseudomonas aeruginosa LSI-2946 4 8 8 0.5

Acinetobacter baumannii LSI-2957 2 1 ≤0.06 0.25

Klebsiella pneumoniae LSI-2999 >64 >64 >64 0.25

Enterobacter cloacae LSI-2943 32 >64 >64 ≤0.06 aDetermined according to CLSI broth microdilution guidelines.34 bVery small pinpoint growth up to 4 µg/mL, no viable growth from pinpoint wells. cPenicillin-susceptible. dPenicillin-resistant. eVancomycin-susceptible. fTrailing observed; no viable growth within trailing wells. gPinpoint growth through 64 µg/mL. hVancomycin-resistant.

3.3.2.2 Targeted enzyme inhibition

The dioxane linked NBTIs described in Chapter 2 were demonstrated to inhibit DNA gyrase more potently than topoisomerase IV. Dual-targeting activity for these new NBTIs (against both DNA gyrase and topoisomerase IV) was one of our major goals, in order to reduce the frequency of spontaneous resistance as in other NBTI projects [7, 23]. To this point, biochemical assays were employed with the new compounds to determine the inhibition of both DNA gyrase and topoisomerase IV, using ciprofloxacin as positive control.

As shown in Table 3.4, our new analogs also inhibited DNA gyrase more potently than topoisomerase IV in general, but critically improved inhibition of topoisomerase IV was obtained for almost all potent new compounds as compared to NBTIs described in Chapter

2. Particularly encouraging were compounds 22 with a topoisomerase IV IC50 = 0.98M and compound 37 with a topoisomerase IV IC50 = 2.8M. They represent promising steps forward on the way to potent dual-targeting compounds. Some compounds displayed less potent topoisomerase IV inhibition using the standard conditions (Method A, see

Experimental Section for details), but substantially improved topoisomerase IV IC50 values were obtained when using more diluted substrates in a slightly modified assay carried out

140 in the Yalowich lab at The Ohio State University (Method B). For instance, compound 25 afforded a topoisomerase IV IC50 >100 M under the standard conditions (Method A) but an IC50 =2.4 M under the modified conditions (Method B). These differences are likely attributable to solubility issues under the conditions of Method A, as the solubility was carefully monitored in Method B. To our delight, almost all new analogs presented more potent topoisomerase IV inhibition than ciprofloxacin using Method B, but efforts are still needed to enhance topoisomerase IV inhibitory activity so as to match DNA gyrase inhibition.

Table 3.4 Target inhibition of new analogs Compound S. aureus S. aureus Compound S. aureus S. aureus DNA gyrase topoisomerase DNA gyrase topoisomerase IC50 (M) IV IC50 (M) IC50 (M) IV IC50 (M) (n=2)a (n=2)b (n=2)a (n=2)b

22 0.03 0.98, 1.0c 38 0.11 8.1

23 0.42 18, 4.3c 39 NYDe 0.6c

24 0.16 5.3, 0.71c 40 0.24 11.0c

25 0.17 >100, 0.18c 44 0.37 11.1c

26 0.13 7.5, 0.28c 57 0.01 0.87c

27 0.04 51.3, 2.4c 58 0.10 2.38c

28 0.07 0.76d 59 0.22 0.99c

29 4.43 >100 60 0.04 2.3c

35 0.02 7.4 61 0.30 5.1c

36 0.16 16.2, 6.8c ciprofloxacin 13.3 4.0, 8.6d

37 0.05 2.8 aSupercoiling inhibition assay, Inspiralis, Ltd. (Norwich, UK). bDecatenation inhibition, Method A (see Experimental Section), determined at Inspiralis (Norwich, UK) (n=2, minimum). cDecatenation inhibition, Method B (see Experimental Section, n=2, minimum). dMethod B (n=1). eNYD=not yet determined 141

In experiments to assess whole cell antibacterial activity, MICs from ciprofloxacin resistant-strains, including USA 300 and SA-3527, consistently demonstrated that these NBTIs lack cross-resistance with fluoroquinolones. Meanwhile, the new NBTIs displayed decreased activity against the 1490-08 strain which has a D83N amino acid substitution in gyrase which is commonly associated with NBTI resistance. Reflecting the whole cell activity back to enzymatic inhibition, these NBTIs are expected to retain enzymatic activity in the presence of mutations that lead to fluoroquinolone resistance [17] but afford diminished inhibition of enzymes with the D83N substitution. Hence, wild-type S. aureus DNA gyrase, S84L mutant gyrase and D83N mutant gyrase were used to test the inhibitory activity of three representative NBTIs (22, 27, 28), with ciprofloxacin as a control.

As shown in Table 3.5, compounds 22 and 27 showed equipotent activity against wild- type and S84L mutant gyrase, while compound 28 showed a modest 4-fold reduction in activity for the S84L gyrase. In contrast, ciprofloxacin lost 16-fold activity against the S84L mutant gyrase. Meanwhile, all the three NBTIs inhibited the D83N mutant gyrase at least 100-fold less potently than the wild-type gyrase, while ciprofloxacin experienced a 44-fold loss of activity with the D83N mutation, consistent with previous literature reports [18]. In summary, NBTIs maintain potent inhibition of DNA gyrase with the classical S84L amino acid substitution but suffer a significant loss of activity against the D83N gyrase mutant. These results from the biochemical assays are fully consistent with whole cell assays in mutant S. aureus strains.

Table 3.5 Inhibition of wild-type and mutant DNA gyrase compound S. aureus wild-type S. aureus S84L S. aureus D83N

a a a DNA gyrase IC50 (M) DNA gyrase IC50 (M) DNA gyrase IC50 (M)

22 0.03 0.018 3.4

27 0.04 0.16 14.4

28 0.07 0.065 8.4

142 ciprofloxacin 13.3 209.9 580.8 aDetermined at Inspiralis (Norwich, UK) (n=2, minimum).

3.3.2.3 Spontaneous Frequency of Resistance

The pursuit of DNA gyrase and topoisomerase IV dual-targeting inhibition was aimed to lower the frequency of resistance (FoR) [24]. To test the spontaneous frequency of resistance for these new analogs, four representative NBTIs (22-24, 27) were assayed in FoR studies using S. aureus ATCC 29213 strain as the test organism and ciprofloxacin as a positive control. FoR values were determined at 4X and 8X multiples of the agar MIC. As presented in Table 3.6, FoRs of the four compounds were in the range of 1x10-7 range at a concentration of 4X MIC with only modest improvements at a concentration of 8X MIC. In contrast, the FoR of Ciprofloxacin was in 10-8 range at 4X MIC and <7.69x10-10 at 8X MIC. Those results were in accordance with the DNA gyrase and topoisomerase IV enzymatic inhibition assays, in which the NBTIs targeted DNA gyrase >10-fold more potently than topoisomerase IV. We hypothesize that further optimization to balance the dual-targeting inhibition is required to substantially decrease the spontaneous frequency of resistance, and additional efforts are ongoing.

Table 3.6 Spontaneous Frequency of Resistance for representative compounds compound S. aureus ATCC Spontaneous Spontaneous

29213 MICb mutation frequency mutation frequency

(g/mL) (n=2) (4X MIC) (8X MIC)

22 0.015 1.53x10-7 4.5x10-8

23 0.03 2.26x10-7 1.08x10-7

24 0.015 6.75x10-7 1.67x10-7

27 0.015 2.31x10-7 1.62x10-7

143

ciprofloxacin 0.25 1.15x10-8 <7.69x10-10 aSee Experimental for full details. bAssays were conducted according to CLSI agar dilution guidelines. [25, 26]

3.3.2.4 Preliminary in vitro safety evaluation

Enhancing cardiovascular safety by reducing hERG inhibition has been a significant objective of modern medicinal chemistry programs [27], especially NBTI projects [28-30]. In Chapter 2, dioxane-linked NBTIs demonstrated that the 5-amino-1,3-dioxane linker was able to reduce hERG inhibition in comparison with other linkers in structure-matched pairs. The new dioxane-linked analogs of Chapter 3 were expected to possess similarly favorable profiles to avert potential cardiac toxicity. All new potent NBTIs, naphthyridine analogs (22-28), (1-hydroxy) naphthyridin-4-yl analogs (35-40), (1-hydroxy) quinolin-4-yl 44 and (2-hydroxy) quinolin-8-yl analogs (57-61), were planned to be evaluated in the hERG inhibition assay, using cisapride as a positive control. Except those latest compounds (58- 61) which have not been determined yet but will be tested in a soon, the resulting data of other new compounds are shown in Table 3.7. Following the same trends as observed with 6-methoxyquinoline analogs in Chapter 2, the pyridothiazinone (25, 37) and pyridooxazinone (26, 38) moieties increased hERG inhibition compared with other enzyme-binding moieties in structure-matched analogs, in conformity with previously reported NBTIs [7, 13]. The (1-hydroxy) naphthyridin-4-yl analogs attained improved hERG profiles over their relevant 6-methoxyquinoline comparators from Chapter 2. For instance, compound 35 afforded hERG IC50 = 27.9 M, a 5-fold reduction of hERG inhibition compared to the structure-matched 6-methoxyquinoline analog (compound 9 in

Chapter 2, hERG IC50 = 5.1 M). Excitingly, some of these new NBTIs achieved very promising hERG IC50 values, achieving our objective for a lead NBTI compound of a hERG IC50 ~100 M [22, 31]. For example, compound 61 possessed a hERG IC50 with 84

M, and compound 36 has a hERG IC50 > 90 M, and compounds 40 and 44 afforded hERG IC50 > 100 M. These results further supported the value of the dioxane linker in terms of reduced hERG inhibition.

144

Table 3.7 In vitro safety assessment of potent NBTIs

b,c Compound hERG IC50 K562 IC50 K/VP.5 hTopoII a (M) (M) IC50 (M)

22 8.0 3.4 3.1 55.0

23 15 68.8 61.8 69.6

24 9.3 53.1 46.0 80.0

25 0.97 25.3 71.9 95.7

26 1.4 >25 >25 83.1

27 7.9 28.9 24.6 94.9

28 13 NYDd NYD 96.4

29 NT 14.0 14.6 90.7

35 >30, 28 52.3 40.6 90.0

36 >90 186.1 232.1 85.7

37 4.8 70.0 52.1 97.9

38 11 187.5 138.5 104.9

39 6.9 NYD NYD NYD

40 >100 NYD NYD NYD

44 >100 >200 106.7 NYD

57 13 NYD NYD NYD

58 40 NYD NYD NYD

59 7.6 NYD NYD NYD

60 NYD NYD NYD NYD

61 84 NYD NYD NYD

Cisapridef 0.012-0.021 NTe NT NT

etoposide NT 0.3 5.1 33.6 (n=4) ciprofloxacin NT NT NT 85.5 (n=4) aDetermined using IonWorks Barracuda at Charles River (Cleveland, OH). bInspiralis, Ltd. (Norwich, UK). cPercent enzyme activity remaining at 100 M. dNYD = not yet determined. eNT = not tested (or not calculated). fCisapride serves as the positive control for the hERG study.

145

As described in Chapter 2, selective inhibition of the bacterial topoisomerases versus the human target by NBTIs is expected due to the sequence and structural distinctions between the eukaryotic and bacterial Type II topoisomerases [32, 33]. Nevertheless, this potential issue was evaluated by testing selected potent NBTIs for selectivity in both indirect and direct assays, as also described in Chapter 2. The indirect assessment of human Topo IIα targeting was completed by the Yalowich group, in which antiproliferative experiments using human leukemia K562 cells and an isogenic (etoposide-resistant) subline, K/VP.5 [34], were conducted. The K/VP.5 cell lines contains 1/5th the level of hTopoII compared to the parental K562 cells. As shown in Table 3.7, K/VP.5 cells demonstrated 17-fold resistance against the positive control etoposide, as compared to K652 cells. The studied NBTIs, in contrast, showed nearly equipotent growth inhibitory

IC50 values against K/VP.5 versus K652 cells, strongly suggesting that these new NBTIs do not target human topoisomerase II. These results were further supported by biochemical assays using isolated human topoisomerase IIα. The human topoisomerase IIα maintained >80% of decatenation activity even at a drug concentration of 100 M, again suggesting that these new compounds are not potent inhibitors of human topoisomerase IIα.

3.4 Conclusions

In this chapter, we sought to take advantage of DNA-binding moieties in previously reported NBTIs, by incorporating them into NBTIs with a 5-amino-1,3-dioxane moiety as the linker domain. N-linked 7-methoxyquinoxalinone analogs (13-16), naphthyridine analogs (22-29), (1-hydroxy) naphthyridin-4-yl analogs (35-40), (1-hydroxy) quinolin-4- yl (44) and (2-hydroxy) quinolin-8-yl (57-61) analogs were all synthesized. With the exception of N-linked quinoxalinone analogs, most new compounds possessed potent whole cell anti-staphylococcal activity, including against fluoroquinolone-resistant strains. The naphthyridine representative (28) showed promising broad-spectrum antibacterial activity and a potent MIC90 against fluoroquinolone-resistant MRSA. In enzyme inhibition assays, these new compounds showed improved topoisomerase IV inhibition and potent DNA gyrase activity. Nevertheless, they inhibited DNA gyrase more potently than 146 topoisomerase IV, leading to a relatively high frequency of spontaneous resistance. Optimization of dual-target inhibition is still needed. Selected NBTIs were assayed for inhibition of two mutant DNA gyrases, one fluoroquinolone-resistant (S84L) and one NBTI-resistant (D83N). The studied NBTIs (22, 27, 28) maintained potent enzymatic activity against the S84L mutant gyrase but lost activity against D83N gyrase, in line with the expected binding mode of NBTIs. Several hydroxy-linked compounds (36, 40, 44) revealed favorable hERG profiles (hERG IC50 ~100 M). Preliminary in vitro human safety assays also illustrated that these new NBTIs displayed low toxicity to human cells and minimal inhibition of human topoisomerase IIα.

In the battle against resistant infections, new classes of antibiotics with distinct mechanism are in demand to increase the arsenal of therapeutics available to human beings. NBTIs represent a promising class as new antibiotics. In this thesis, a 5-amino-1,3-dioxane moiety was used as the linker in new NBTIs and demonstrated to reduce hERG inhibition. A variety of 5-amino-1,3-dioxane linked compounds have been synthesized and evaluated in in vitro studies as new NBTIs. Several newly synthesized compounds possessed excellent preclinical attributes, such as compound 36 and 40 in Chapter 3. These promising compounds have the potential to be evaluated in in vivo experiments and can serve as the starting point for further development of new NBTIs. Hopefully this work can help future studies in our group or inspire other researchers with additional innovative projects on NBTIs.

3.5 Experimental Section

3.5.1 Chemistry part

147 were recorded at either 300 MHz or 400 MHz using residual protiated solvent as the internal reference: CDCl3 (7.26 ppm), CD3OD (3.31), DMSO-d6 (2.50 ppm). Assayed compounds had a purity of >90% as determined by 1H NMR analysis. 13C NMR spectra were recorded at 75 MHz or 100 MHz using the solvent signal as the internal reference:

CDCl3 (77.16 ppm), CD3OD (49.00), DMSO-d6 (39.52 ppm). High resolution mass spectrometry was performed using electrospray ionization. The equatorial protons at C4 and C6 of the trans-dioxane ring typically appear as an apparent doublet of doublets at ca. 4.1-4.2 ppm in the analogs described below.[35] Careful inspection in some cases reveals additional partially resolved splitting, arising from W-coupling of these magnetically non- equivalent protons as well as virtual coupling. The additional splitting can be effectively replicated using computer simulation of non-first order effects [36], using a W-coupling constant of 1.8 Hz. For the sake of clarity and consistency, this peak is labeled a doublet of doublets in the characterization data provided below.

3-(benzyloxy)propanal (2). To a solution of 3-(benzyloxy)propan-1-ol (857 mg, 5.16 mmol, 1.0 equiv.) in DCM (10 mL) was added Dess-Martin periodinane (2.4g, 5.7 mmol, 1.1 equiv.) at 0 °C. The reaction mixture was stirred at room temperature for 3 hours. Saturated aqueous sodium thiosulfate (10 mL) and saturated aqueous sodium bicarbonate (10 mL) were added and the mixture stirred for 30 minutes. The reaction mixture was filtered through Celite and phases separated. The organic layer was washed twice with brine, then concentrated to give the title compound as a colorless oil (780 mg, 4.75 mmol, 90% yield), used without further purification.

148

2-(2-(2-(benzyloxy)ethyl)-trans-1,3-dioxan-5-yl)isoindoline-1,3-dione (4). A mixture of compound 2 (83.7 mg, 0.51 mmol, 1.0 equiv), compound 3 (110 mg, 0.50 mmol, 1.0 equiv), p-toluenesulfonic acid (2 mg), 4Å molecular sieves (20 mg), and toluene (4 mL) was stirred and heated at 110 °C under N2 atmosphere overnight. The mixture was filtered, and the solution was then washed by saturated aqueous NaHCO3 and extracted with DCM. The combined organic layers were concentrated, and the crude product was purified by chromatography on silica gel with hexane/ethyl acetate (4:1) to afford the title compound 1 as a white solid (122 mg, 0.332 mmol, 67% yield). H NMR (300 MHz, CDCl3) δ: 7.87- 7.83 (m, 2H); 7.77-7.74 (m, 2H); 7.36-7.28 (m, 5H); 4.85 (t, J=5.2 Hz, 1H); 4.71-4.58 (m, 1H); 4.55 (s, 2H); 4.43 (t, J=10.6 Hz, 2H); 4.01 (dd, J=10.5, 4.7 Hz, 2H); 3.61 (t, J=6.4 Hz, 13 2H); 1.99 (dt, J=6.4, 5.2 Hz, 2H). C NMR (75 MHz, CDCl3) δ: 168.0, 138.6, 134.4, 131.8, 128.5, 127.8, 127.7, 123.6, 99.9, 73.2, 66.4, 65.7, 44.4, 35.1.

2-(2-(2-hydroxyethyl)-trans-1,3-dioxan-5-yl)isoindoline-1,3-dione (5). Compound 4 (183.7 mg, 0.50 mmol) was dissolved in methanol (3mL), treated with 10% palladium on carbon (15 mg), and stirred under an atmosphere of hydrogen overnight. The reaction mixture was filtered through Celite, and the filtrate was concentrated to afford the title 1 compound as a white solid (123.4 mg, 0.445 mmol, 89% yield). H NMR (300 MHz, CDCl3) δ: 7.90-7.78 (m, 2H); 7.78-7.64 (m, 2H); 4.89 (t, J=4.8 Hz, 1H); 4.70-4.54 (m, 1H); 4.43 (t, J=10.2 Hz, 2H); 4.04 (dd, J=10.6, 4.7 Hz, 2H); 3.80 (br s, 2H); 2.33 (brs, 1H); 1.97-1.92 13 (m, 2H). C NMR (75 MHz, CDCl3) δ: 167.9, 134.5, 131.7, 123.6, 101.5, 66.4, 58.7, 44.2, 36.8.

149

2-(5-(1,3-dioxoisoindolin-2-yl)-trans-1,3-dioxan-2-yl)ethyl methanesulfonate (6). Compound 5 (138.7 mg, 0.50 mmol, 1.0 equiv) was dissolved in pyridine (1mL), methanesulfonic anhydride (143.7 mg, 0.82 mmol, 1.6 equiv) was added at 0 °C, and then the mixture was stirred at room temperature for 4 hours. Ethyl acetate (10 mL) and brine (5 mL) were added and the phases separated. The aqueous layer was extracted with ethyl acetate (10 mL), and the combined organic layers were concentrated to provide the crude product which was purified by chromatography on silica gel with DCM/methanol (100:1) to afford the title compound as a white solid as product (144 mg, 0.405 mmol, 81% yield). 1 H NMR (300 MHz, CDCl3) δ: 7.85-7.81 (m, 2H); 7.77-7.72 (m, 2H); 4.84 (t, J=4.9 Hz, 1H); 4.68-4.53 (m, 1H); 4.50-4.29 (m, 4H); 4.03 (dd, J=10.4, 4.6 Hz, 2H); 3.03 (s, 3H); 13 2.12 (dt, J=6.4, 4.9 Hz, 2H). C NMR (75 MHz, CDCl3) δ: 167.9, 134.5, 131.7, 123.6, + 98.5, 66.4, 65.8, 44.2, 37.5, 34.3. HRMS (ESI) m/z calc’d for C15H17NO7SNa [M+Na] : 378.0623; found: 378.0608.

Ethyl N-(4-Methoxy-2-nitrophenyl)glycinate (8). A mixture of 4-methoxy-2-nitroaniline 7 (500 mg, 2.91 mmol, 1.0 equiv.), ethyl bromoacetate (4 mL, 35.35 mmol, 35.3 equiv.), and potassium carbonate (804 mg, 5.82 mmol, 2.0 equiv.) was heated at 150 °C for 4.5 h. The mixture was cooled to room temperature, and aqueous sodium hydroxide solution (1M, 12 mL) was added. This mixture was extracted with DCM. The combined organic phases were dried over magnesium sulfate and concentrated under reduced pressure. Chromatography was done on silica gel with hexanes/ethyl acetate (5:1) to give target 1 product (588 mg, 2.31 mmol, 79%) as a red solid. H NMR (300 MHz, CDCl3) δ: 7.91 (brs,

150

1H); 7.62 (d, J=3.0 Hz, 1H); 7.15 (dd, J=9.4, 3.0 Hz, 1H); 6.82 (d, J=9.4 Hz, 1H); 3.79 (s, 3H); 3.35 (qd, J=7.2, 5.2 Hz, 2H); 1.36 (t, J=7.2 Hz, 3H).

7-Methoxy-3,4-dihydroquinoxalin-2(1H)-one (9). Compound 8 (588 mg, 2.31 mmol, 1.0 equiv.) was taken up in 7.5 mL of 1:1 methanol/acetic acid, treated with 10% palladium on carbon (75 mg), and stirred in an atmosphere of hydrogen overnight. The reaction mixture was filtered through Celite and the filtrate was concentrated as a tan solid (340 mg), used without further purification.

7-Methoxyquinoxalin-2(1H)-one (10). To a solution of 8% aqueous sodium hydroxide (4.5 mL) was added intermediate 9 (340 mg) followed by a solution of 30 wt % hydrogen peroxide in water (1.97 mL). The reaction mixture was slowly heated to 80 °C and maintained at this temperature for 4 h. The mixture was cooled down to room temperature, and acetic acid (510 μL) was added dropwise. The suspension was stirred overnight at room temperature and the precipitated solid was collected by filtration to afford the product as a 1 tan solid (208 mg, 1.18 mmol, 51% for two steps). H NMR (300 MHz, DMSO-d6) δ: 12.30 (brs, 1H); 7.97 (s, 1H); 7.68 (d, J=8.9 Hz, 1H); 6.91 (dd, J=8.9, 2.7 Hz, 1H); 6.76 (d, J=2.7 Hz, 1H); 3.83 (s, 3H).

2-(2-(2-(7-methoxy-2-oxoquinoxalin-1(2H)-yl)ethyl)-trans-1,3-dioxan-5-

151 yl)isoindoline-1,3-dione (11). To a solution of compound 10 (88.1 mg, 0.50 mmol, 1.0 equiv) in DMSO (2 mL) was added cesium carbonate (325.8mg, 1.0 mmol, 2.0 equiv) at 0 °C. After 30 minutes, compound 6 (177.7 mg, 0.50 mmol, 1.0 equiv) was added and stirred overnight. The reaction mixture was treated with brine (20 mL) and extracted with DCM. The combined organic layers were concentrated, and the crude product was purified by chromatography on silica gel with hexane/ethyl acetate (3:1) to give the title compound 1 as a white solid (78.4 mg, 0.180 mmol, 36%). H NMR (300 MHz, CDCl3) δ: 8.33 (s, 1H); 7.89-7.80 (m, 3H); 7.76-7.69 (m, 2H); 7.22-7.15 (m, 2H); 4.96 (t, J=5.1 Hz, 1H); 4.72-4.60 (m, 3H); 4.45 (t, J=10.7 Hz, 2H); 4.06 (dd, J=10.7, 4.7 Hz, 2H); 3.94 (s, 3H); 2.24 (dt, 13 J=6.4, 5.1 Hz, 2H). C NMR (75 MHz, CDCl3) δ: 168.0, 161.2, 157.9, 142.3, 136.6, 134.7, 134.4, 131.7, 130.0, 123.6, 118.7, 106.2, 99.5, 66.5, 62.1, 55.9, 44.4, 34.1.

1-(2-(5-amino-trans-1,3-dioxan-2-yl)ethyl)-7-methoxyquinoxalin-2(1H)-one (12). A mixture of Compound 11 (217.8 mg, 0.50 mmol. 1.0 equiv), ethanolamine (46 μL, 7.6mmol, 15 equiv) and ethyl acetate (4 mL) was stirred and heated at 70 °C overnight. The solvent was removed, and the mixture was partitioned between DCM and brine and phases separated. The aqueous layer was extracted with DCM, and the combined organic layers were concentrated. The crude product was purified by chromatography on silica gel with DCM/methanol (15:1) to give the title compound as an oil (116 mg, 0.380 mmol, 76% 1 yield). H NMR (300 MHz, CDCl3) δ: 8.29 (s, 1H); 7.85 (d, J = 8.8 Hz, 1H); 7.19-7.14 (m, 2H); 4.67 (t, J=5.2 Hz, 1H); 4.55 (t, J=6.5 Hz, 2H); 4.13 (dd, J=11.0, 4.7 Hz, 2H); 3.92 (s, 3H); 3.25 (t, J=10.8 Hz, 2H); 3.13-3.00 (m, 1H); 2.17 (dt, J=6.5, 5.2 Hz, 2H). 13C NMR

(75 MHz, CDCl3) δ: 161.2, 157.8, 142.3, 136.6, 134.6, 129.9, 118.7, 106.2, 99.2, 73.6, 62.2, + 55.8, 44.3, 34.1. HRMS (ESI) m/z calc’d for C15H19N3O4Na [M+Na] : 328.1273; found: 328.1272.

Reductive amination general procedure for 13-16: To a solution of primary amine 12 152

(0.2 mmol) in methanol (2 mL) was added the requisite aldehyde (0.2 mmol) and zinc chloride (2 mg, 0.015 mmol, 0.07 equiv). The mixture was stirred at room temperature for 30 min, followed by addition of sodium cyanoborohydride (40 mg, 0.6 mmol, 3.0 equiv). The reaction mixture was stirred at room temperature overnight and then purified by chromatography on silica gel with DCM/methanol (50:1).

1-(2-(5-(((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)amino)-trans-1,3-dioxan-2- yl)ethyl)-7-methoxyquinoxalin-2(1H)-one (13). The title compound was obtained as a 1 colorless oil in 44 % yield following the general method. H NMR (300 MHz, CDCl3) δ 8.29 (s, 1H); 7.86 (d, J=8.9 Hz, 1H); 7.18-7.14 (m, 2H); 6.81-6.72 (m, 3H); 4.68 (t, J=5.2 Hz, 1H); 4.55 (t, J=6.5 Hz, 2H); 4.23 (s, 4H); 4.18 (dd, J=11.3, 4.7 Hz, 2H); 3.93 (s, 3H); 3.68 (s, 2H); 3.31 (t, J=10.9 Hz, 2H); 2.99-2.94 (m, 1H); 2.16 (dt, J=6.5, 5.2 Hz, 2H). 13C

NMR (75 MHz, CDCl3) δ 161.2, 157.9, 143.6, 142.9, 142.3, 136.6, 134.6, 133.6, 129.9, 121.0, 118.7, 117.4, 116.9, 106.2, 99.5, 71.8, 64.49, 64.45, 62.2, 55.8, 50.9, 49.8, 34.2. + HRMS (ESI) m/z calc’d for C24H28N3O6 [M+H] : 454.1978; found: 454.1972.

1-(2-(5-(((2,3-dihydro-[1,4]dioxino[2,3-c]pyridin-7-yl)methyl)amino)-trans-1,3- dioxan-2-yl)ethyl)-7-methoxyquinoxalin-2(1H)-one (14). The title compound was obtained as a yellow oil in 29 % yield following the general method. 1H NMR (300 MHz,

CDCl3) δ 8.29 (s, 1H); 8.09 (s, 1H); 7.86 (d, J=8.7 Hz, 1H); 7.19-7.15 (m, 2H); 6.77 (s, 1H); 4.69(t, J=5.2 Hz, 1H); 4.55 (t, J=6.5 Hz, 2H); 4.33-4.25 (m, 4H); 4.19 (dd, J=11.3, 4.7 Hz, 2H); 3.93 (s, 3H); 3.77 (s, 2H); 3.36 (t, J=10.9 Hz, 2H); 3.00-2.91 (m, 1H); 2.16 13 (dt, J=6.5, 5.2 Hz, 2H). C NMR (75 MHz, CDCl3) δ 161.2, 157.9, 153.1, 150.4, 142.3,

153

140.4, 139.1, 136.6, 134.7, 130.0, 118.7, 110.8, 106.2, 99.5, 71.7, 65.1, 64.2, 62.2, 55.9, + 52.2, 50.2, 34.3. HRMS (ESI) m/z calc’d for C23H26N4O6Na [M+Na] : 477.1750; found: 477.1740.

1-(2-(5-(((3,4-dihydro-2H-pyrano[2,3-c]pyridin-6-yl)methyl)amino)-trans-1,3- dioxan-2-yl)ethyl)-7-methoxyquinoxalin-2(1H)-one (15). The title compound was 1 obtained as a film in 80 % yield following the general method. H NMR (300 MHz, CDCl3) δ: 8.30(s, 1H); 8.08 (s, 1H); 7.86 (d, J=9.1 Hz, 1H); 7.20-7.15 (m, 2H); 6.92 (s, 1H); 4.70 (t, J=5.2 Hz, 1H); 4.55 (t, J=6.5 Hz, 2H); 4.24-4.18 (m, 4H); 3.93 (s, 3H); 3.79 (s, 2H); 3.38 (t, J=10.9 Hz, 2H); 3.01-2.97 (m, 1H); 2.75 (t, J=6.6 Hz, 2H); 2.16 (dt, J=6.5, 5.2 Hz, 13 2H); 2.07-1.96 (m, 2H). C NMR (75 MHz, CDCl3) δ: 161.3, 157.9, 151.2, 150.1, 142.3, 139.0, 136.7, 134.7, 131.2, 130.0, 122.8, 118.7, 106.3, 99.6, 71.7, 66.7, 62.2, 55.9, 52.1, + 50.2, 34.3, 24.4, 21.8. HRMS (ESI) m/z calc’d for C24H28N4O5Na [M+Na] : 475.1957; found: 475.1953.

1-(2-(5-((3,4-dichlorobenzyl)amino)-trans-1,3-dioxan-2-yl)ethyl)-7- methoxyquinoxalin-2(1H)-one (16). The title compound was obtained as a tan solid in 1 53 % yield following the general method. H NMR (300 MHz, CDCl3) δ: 8.30 (s, 1H); 7.87 (d, J=8.8 Hz, 1H); 7.43-7.37 (m, 2H); 7.21-7.13 (m, 3H); 4.70 (t, J=5.2 Hz, 1H); 4.55 (t, J=6.5 Hz, 2H); 4.20 (dd, J=11.2, 4.7 Hz, 2H); 3.94 (s, 3H); 3.78 (s, 2H); 3.33 (t, J=10.8 13 Hz, 2H); 3.00-2.91 (m, 1H); 2.17 (dt, J=6.5, 5.2 Hz, 2H). C NMR (75 MHz, CDCl3) δ:161.3, 157.9, 142.3, 136.6, 134.7, 132.7, 130.6, 130.0, 127.4, 118.7, 106.2, 99.6, 71.5, + 62.1, 55.9, 50.3, 50.0, 34.2. HRMS (ESI) m/z calc’d for C22H24Cl2N3O4 [M+H] : 464.1144; 154 found: 464.1146.

2-(2-vinyl-trans-1,3-dioxan-5-yl)isoindoline-1,3-dione (17). A mixture of acrolein (4.16 mL, 62.3 mmol, 3.1 equiv.), diol 3 (4.426 g, 20.01 mmol, 1.0 equiv.), p-toluenesulfonic acid (0.12 g, 0.63 mmol, 0.03 equiv.), anhydrous cupric sulfate (1.6 g, 10. mmol, 0.5 equiv.) and anhydrous DCM (60 mL) was stirred at room temperature under N2 atmosphere for 36 h. The mixture was then washed with brine, and the aqueous phase was extracted with DCM. The combined organic layers were concentrated, and the crude product was purified by chromatography on silica gel with DCM/methanol (50:1) to give the title compound as 1 a white solid (3.948 g, 15.23 mmol, 76%). H NMR (300 MHz, CDCl3) δ: 7.88-7.81 (m, 2H); 7.77-7.70 (m, 2H) 5.90 (ddd, J = 4.5, 10.7, 17.4, 1H); 5.53 (d apparent t, J = 1.2, 17.4, 1H); 5.36 (d apparent t, J = 1.1, 10.7, 1H); 5.10 (br d, J = 4.5, 1H); 4.73-4.61 (m, 1H); 4.55-4.46 (m, 2H); 4.09 (dd, J = 4.7, 10.6, 2H).

8-bromo-7-fluoro-2-methoxy-1,5-naphthyridine (19). To a solution of commercial 3- fluoro-6-methoxy-1,5-naphthyridin-4-ol 18 (0.8625 g, 4.442 mmol, 1.0 equiv.) in DMF (10 mL) was dropped phosphorus tribromide (466 μL, 5.0 mmol, 1.12 equiv.) at 0 °C under N2 atmosphere. The reaction mixture was stirred for 1 hour, then water (200 mL) and aqueous sodium hydroxide solution (6N, 740 μL) were added. The reaction mixture was stirred for 2 hours, and the precipitated solid was collected by filtration to afford the title compound 1 as a tan solid (1.03 g, 4.01 mmol, 90% yield). H NMR (300 MHz, CDCl3) δ: 8.62 (s, 1H); 8.21 (d, J = 9.0 Hz, 1H); 7.13 (d, J = 9.0 Hz, 1H); 4.17 (s, 3H).

155

2-(2-(2-(3-fluoro-6-methoxy-1,5-naphthyridin-4-yl)ethyl)-trans-1,3-dioxan-5- yl)isoindoline-1,3-dione (20). To a solution of intermediate 17 (155.6 mg, 0.60 mmol, 1.2 equiv relative to compound 19) in THF (3 mL) was added dropwise 9- borabicyclo[3.3.1]nonane solution (1.2 mL, 0.5 M in THF, 0.60 mmol, 1.0 equiv relative to 17) at room temperature under N2 atmosphere. The mixture was stirred for 3 hours and used directly later. To a mixture of 19 (128.5 mg, 0.50 mmol, 1.0 equiv), cesium carbonate (325.8 mg, 1.00 mmol), and [1,1`-bis(diphenylphosphino)ferrocene] dichloropalladium(II) (13.4 mg, 0.0183 mmol, 0.04 equiv) in THF (3 mL) was added dropwise the fresh solution described above at room temperature under N2 atmosphere. The reaction mixture was stirred overnight. The solvent was then removed in vacuo, and the residue was dissolved in DCM and washed with brine. The organic layer was concentrated and purified by chromatography on silica gel with hexane/ethyl acetate (3:1) to give the title compound as 1 a white solid (126.4 mg, 0.29 mmol, 58% yield). H NMR (300 MHz, CDCl3) δ: 8.61 (s, 1H); 8.16 (d, J = 9.0 Hz, 1H); 7.84-7.80 (m, 2H); 7.77-7.68 (m, 2H); 7.06 (d, J = 9.0 Hz, 1H); 4.73 (t, J = 5.0 Hz, 1H); 4.69-4.54 (m, 1H); 4.39 (t, J = 10.6 Hz, 2H); 4.11 (s, 3H); 4.02 (dd, J = 10.7, 4.8 Hz, 2H); 3.32 (t, J = 7.3 Hz, 2H); 2.13 (td, J = 7.7, 5.2 Hz, 2H).

2-(2-(3-fluoro-6-methoxy-1,5-naphthyridin-4-yl)ethyl)-trans-1,3-dioxan-5-amine (21). A mixture of 20 (218.7 mg, 0.50 mmol, 1.0 equiv), ethanolamine (46 μL, 7.6 mmol, 15 equiv.) and ethyl acetate (4 mL) was stirred and heated at 70 °C overnight. The solvent was removed, and the residue was dissolved in DCM and washed with brine. The organic layer concentrated, and the crude product was purified by chromatography on silica gel 156 with DCM/methanol (15:1) to give the title compound as an oil (123.1 mg, 0.40 mmol, 80% 1 yield). H NMR (CDCl3) δ: 8.58 (s, 1H); 8.14 (d, J = 9.0 Hz, 1H); 7.03 (d, J = 9.0 Hz, 1H); 4.41 (t, J = 5.1 Hz, 1H); 4.14-4.06 (m, 5H); 3.26-3.14 (m, 4H); 3.08-3.00 (m, 1H); 2.09- 1.97 (m, 2H); 1.09 (br s, 2H).

Reductive amination general procedure for 22-29: To a solution of amine 21 (0.2 mmol) in methanol (2 mL) was added the requisite aldehyde (0.2 mmol) and zinc chloride (2 mg, 0.015 mmol, 0.07 equiv). The mixture was stirred at room temperature for 30 min, followed by addition of sodium cyanoborohydride (40 mg, 0.6 mmol, 3.0 equiv). The reaction mixture was stirred at room temperature overnight and then purified by chromatography on silica gel with DCM/methanol (50:1). On occasion, the product obtained from flash 1 chromatography was contaminated by a BH3 adduct, seen in the H NMR spectrum as four broad peaks from 0.00-0.88 ppm. In these instances, the column-purified material was dissolved in methanol (2 mL) and stirred overnight at ambient temperature with Amberlite IRA743 free base (ca. 100 mg). The pure title compound was then obtained by removal of the resin by filtration and removal of the solvent under reduced pressure.

O O N O H O N F O

N 22

N-((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)-2-(2-(3-fluoro-6-methoxy-1,5- naphthyridin-4-yl)ethyl)-trans-1,3-dioxan-5-amine (22). The title compound was prepared in 52% yield following the general method and obtained as a white solid. 1H NMR

(300 MHz, CDCl3) δ: 8.59 (s, 1H); 8.15(d, J=9.0 Hz, 1H); 7.04 (d, J=9.0 Hz, 1H); 6.71- 6.81 (m, 3H); 4.44 (t, J=5.0 Hz, 1H); 4.23 (s, 4H); 4.15 (dd, J=11.1, 4.7 Hz, 2H); 4.07 (s, 3H); 3.67 (s, 2H); 3.21-3.27 (m, 4H); 2.90-2.95 (m, 1H); 2.05 (td, J=7.6, 5.2 Hz, 2H);. 13C

NMR (75 MHz, CDCl3) δ: 162.4, 158.9, 155.5, 143.6, 142.9, 141.74, 141.65, 140.2, 138.62, 138.59, 138.2, 137.8, 133.6, 131.9, 131.7, 121.0, 117.4, 116.9, 115.24, 115.20, 101.7, 71.7,

64.49, 64.46, 53.9, 50.9, 49.7, 33.6, 18.4. HRMS (ESI) m/z calc’d for C24H27FN3O5 [M+H]+: 456.1934; found: 456.1910. 157

O O N H O N O N F O

N 23

N-((2,3-dihydro-[1,4]dioxino[2,3-c]pyridin-7-yl)methyl)-2-(2-(3-fluoro-6-methoxy- 1,5-naphthyridin-4-yl)ethyl)-trans-1,3-dioxan-5-amine (23). The title compound was prepared in 43% yield following the general method and obtained as a thin film. 1H NMR

(300 MHz, CDCl3) δ: 8.58 (s, 1H); 8.14 (d, J=9.0 Hz, 1H); 8.08 (s, 1H); 7.04 (d, J=9.0 Hz, 1H); 6.75 (s, 1H); 4.44 (t, J=5.0 Hz, 1H); 4.33-4.23 (m, 4H); 4.15 (dd, J=11.2, 4.7 Hz, 2H); 4.06 (s, 3H); 3.75 (s, 2H); 3.35-3.22(m, 4H); 3.00-2.86 (m, 1H); 2.04 (td, J=7.6, 5.2 Hz, 13 2H). C NMR (75 MHz, CDCl3) δ: 162.4, 158.9, 155.5, 153.1, 150.4, 141.7, 141.6, 140.3, 140.2, 139.0, 138.59, 138.57, 138.2, 137.8, 131.9, 131.8, 115.25, 115.21, 110.8, 101.7, 71.7, + 65.1, 64.2, 53.9, 52.1, 50.0, 33.6, 18.4. HRMS (ESI) m/z calc’d for C23H26FN4O5 [M+H] : 457.1887; found: 457.1871.

O N H O N O N F O

N 24

N-((3,4-dihydro-2H-pyrano[2,3-c]pyridin-6-yl)methyl)-2-(2-(3-fluoro-6-methoxy-1,5- naphthyridin-4-yl)ethyl)-trans-1,3-dioxan-5-amine (24). The title compound was prepared in 77% yield following the general method and obtained as a thin film. 1H NMR

(300 MHz,CDCl3) δ: 8.58 (s, 1H); 8.14 (d, J=9.0 Hz, 1H); 8.05 (s, 1H); 7.04 (d, J=9.0 Hz, 1H); 6.95 (s, 1H); 4.47 (t, J=5.0 Hz, 1H); 4.22-4.15 (m, 4H); 4.07 (s, 3H); 3.81 (s, 2H); 3.37 (t, J=10.8 Hz, 2H); 3.26 (t, J=7.6 Hz, 2H); 3.02-2.95 (m, 1H); 2.76 (t, J=6.5 Hz, 2H); 13 2.08-2.00 (m, 4H). C NMR (75 MHz, CDCl3) δ: 162.5, 158.9, 155.5, 151.5, 149.0, 141.7, 141.6, 140.2, 138.62, 138.59, 138.2, 137.8, 131.9, 131.7, 123.1, 115.27, 115.23, 101.8, 71.1,

66.8, 53.9, 51.5, 50.2, 33.5, 24.4, 21.6, 18.4. HRMS (ESI) m/z calc’d for C24H27FN4O4 [M+Na]+: 477.1914; found: 477.1895.

158

6-(((2-(2-(3-fluoro-6-methoxy-1,5-naphthyridin-4-yl)ethyl)-trans-1,3-dioxan-5- yl)amino)methyl)-2H-pyrido[3,2-b][1,4]thiazin-3(4H)-one (25). The title compound was prepared in 44 % yield following the general method as a yellow solid. 1H NMR

(300 MHz, CDCl3) δ: 8.61 (s, 1H); 8.60 (s, 1H); 8.16 (d, J=9.0 Hz, 1H); 7.57 (d, J=7.8 Hz, 1H); 7.05 (d, J=9.0 Hz, 1H); 6.94 (d, J=7.8 Hz, 1H); 4.45 (t, J=5.0 Hz, 1H); 4.17 (dd, J=11.2, 4.7 Hz, 2H); 4.07 (s, 3H); 3.82 (s, 2H); 3.47 (s, 2H); 3.35-3.24 (m, 4H); 2.94 (tt, 13 J=9.8, 4.7 Hz, 1H); 2.06 (td, J=7.6, 5.2 Hz, 2H). C NMR (75 MHz, CDCl3) δ: 165.7, 162.5, 158.9, 156.8, 155.5, 148.4, 141.8, 141.7, 140.3, 138.6, 138.2, 137.8, 136.4, 131.9, 131.7, 117.9, 115.28, 115.24, 114.0, 101.8, 71.6, 53.9, 51.7, 50.1, 33.6, 29.8, 18.4. HRMS + (ESI) m/z calc’d for C23H25FN5O4S [M+H] : 486.1611; found: 486.1596.

6-(((2-(2-(3-fluoro-6-methoxy-1,5-naphthyridin-4-yl)ethyl)-trans-1,3-dioxan-5- yl)amino)methyl)-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one (26). The title compound was prepared in 43 % yield following the general method as a white solid. 1H NMR (300

MHz, CDCl3) δ: 8.60 (s, 1H); 8.17 (d, J=9.0 Hz, 1H); 7.21 (d, J=8.0 Hz, 1H); 7.06 (d, J=9.0 Hz, 1H); 6.91 (d, J=8.1 Hz, 1H); 4.65 (s, 2H); 4.47 (t, J=5.0 Hz, 1H); 4.18 (dd, J=11.2, 4.7 Hz, 2H); 4.08 (s, 3H); 3.81 (s, 2H); 3.57 (brs, 1H); 3.39-3.24(m, 4H); 3.01- 13 2.92 (m, 1H); 2.06 (td, J=7.5, 5.3 Hz, 2H). C NMR (75 MHz, CDCl3) δ: 165.4, 162.5, 158.9, 155.5, 141.8, 141.7, 140.3, 138.7, 138.5, 138.2, 137.9, 131.7, 124.4, 118.3, 115.3, 101.8, 71.4, 67.4, 53.9, 51.5, 50.1, 33.6, 29.9, 18.4. HRMS (ESI) m/z calc’d for + C23H25FN5O5 [M+H] : 470.1840; found: 470.1821.

159

N-(3,4-dichlorobenzyl)-2-(2-(3-fluoro-6-methoxy-1,5-naphthyridin-4-yl)ethyl)-trans- 1,3-dioxan-5-amine (27). The title compound was prepared in 45% yield following the 1 general method and obtained as a white solid. H NMR (300 MHz, CDCl3) δ: 8.60 (s, 1H); 8.16 (d, J=9.0 Hz, 1H); 7.37-7.42 (m, 2H); 7.13 (dd, J=8.2, 1.8 Hz, 1H); 7.06 (d, J=9.0 Hz, 1H); 4.45 (t, J=5.0 Hz, 1H); 4.16 (dd, J=11.2, 4.7 Hz, 2H); 4.08 (s, 3H); 3.76 (s, 2H) 3.24- 13 3.35 (m, 4H); 2.88-2.95 (m, 1H); 2.07 (td, J=7.6, 5.3 Hz, 2H);. C NMR (75 MHz, CDCl3) δ: 162.5, 158.9, 155.5, 141.8, 141.7, 140.6, 140.3, 138.67, 138.64, 138.2, 137.9, 132.7, 131.9, 131.7, 131.3, 130.6, 130.0, 127.3, 115.30, 115.26, 101.8, 71.6, 53.9, 50.3, 50.0, 33.6, + 18.4. HRMS (ESI) m/z calc’d for C22H23Cl2FN3O3 [M+H] : 466.1100; found: 466.1088.

O S N H O N O N F N O

N 28

N-((6,7-dihydro-[1,4]oxathiino[2,3-c]pyridazin-3-yl)methyl)-2-(2-(3-fluoro-6- methoxy-1,5-naphthyridin-4-yl)ethyl)-trans-1,3-dioxan-5-amine (28). The title compound was prepared in 58 % yield following the general method and obtained as light 1 yellow solid. H NMR (300 MHz, CDCl3) δ 8.58 (s, 1H); 8.14 (d, J=9.0 Hz, 1H); 7.27 (s, 1H); 7.04 (d, J=9.0 Hz, 1H); 4.63 (t, J=4.7 Hz, 2H); 4.46 (t, J=4.8 Hz, 1H); 4.20 (dd, J=11.1, 4.7 Hz, 2H); 4.07 (s, 3H); 3.96 (s, 2H); 3.38-3.22 (m, 4H); 3.19 (t, J=4.7 Hz, 2H); 3.03- 13 2.90 (m, 1H); 2.07-2.01 (m, 2H). C NMR (75 MHz, CDCl3) δ: 162.4, 160.1, 158.9, 156.0, 155.5, 141.7, 141.6, 140.2, 138.61, 138.59, 138.2, 137.8, 131.8, 131.7, 126.0, 125.2, 115.25, 115.22, 101.7, 71.2, 66.3, 53.9, 50.12, 50.09, 33.5, 25.7, 18.4. HRMS (ESI) m/z calc’d for + C22H25FN5O4S [M+H] : 474.1611; found: 474.1614.

160

N-(3,4-difluorobenzyl)-2-(2-(3-fluoro-6-methoxy-1,5-naphthyridin-4-yl)ethyl)-trans- 1,3-dioxan-5-amine (29). The title compound was prepared in 43% yield following the 1 general method and obtained as a white solid. H NMR (400 MHz, CDCl3) δ 8.59 (s, 1H); 8.15 (d, J=9.0 Hz, 1H); 7.18-7.03 (m, 3H); 7.02-6.97 (m, 1H); 4.44 (t, J=5.1 Hz, 1H); 4.18- 4.13 (m, 2H); 4.07 (s, 3H); 3.75 (s, 2H); 3.30-3.22(m, 4H); 2.97-2.89 (m, 1H); 2.11-2.02 13 (m, 2H). C NMR (100 MHz, CDCl3) δ 162.5, 158.5, 155.9, 151.8, 151.7, 150.9, 150.8, 149.3, 149.2, 148.5, 148.4, 141.73, 141.66, 140.2, 138.63, 138.61, 138.2, 137.9, 137.46, 137.41, 137.37, 131.8, 131.7, 123.77, 123.74, 123.71, 123.67, 117.3, 117.2, 116.9, 116.7, 115.28, 115.26, 101.8, 71.6, 53.9, 50.4, 49.9, 33.5, 18.4. HRMS (ESI) m/z calc’d for + C22H23F3N3O3 [M+H] : 434.1691; found: 434.1682.

2-(2-(2,2-dimethoxyethyl)-1,3-dioxan-5-yl)isoindoline-1,3-dione (31). 2-(1,3- dihydroxypropan-2-yl)isoindoline-1,3-dione 3 (5.61 g, 25.37 mmol, 1.0 equiv.) and commercial 1,1,3,3-tetramethoxypropane 30 (5 g, 30.45 mmol, 1.2 equiv.) were suspended in toulune (50 mL). p-TsOH (970 mg, 5.07 mmol, 0.20 equiv.) was added, the mixture was heated to 85 °C with a Dean–Stark apparatus. The mixture lasted around 4 hours and then cooled to room temperature. Chromatography was done on silica gel with hexane/ethyl acetate (3:1) to give product as a white solid (2.13 g, 6.53 mmol, 26%). 1H NMR (300

MHz, CDCl3) δ: 7.84 (dd, J = 5.6, 3.0 Hz, 2H); 7.73 (dd, J = 5.6, 3.0 Hz, 2H); 4.77 (t, J = 5.4 Hz, 1H); 4.69-4.53 (m, 2H); 4.43 (t, J = 10.3 Hz, 2H); 4.02 (dd, J = 10.5, 4.8 Hz, 2H); 3.36 (s, 6H); 2.00 (t, J = 5.7Hz, 2H).

161

2-(5-(1,3-dioxoisoindolin-2-yl)-1,3-dioxan-2-yl)acetaldehyde (32). Compound 31 (2.13 g, 6.53 mmol, 1.0 equiv.) was dissolved in THF (12 mL) and water (48 mL). Then PPTS (164.2 mg, 0.653 mmol, 0.1 equiv.) was added and the mixture was heated to 80 °C. The reaction mixture lasted 10 hours and then extracted by DCM. Chromatography was done on silica gel with methanol/DCM (1:50) to give product as a white solid (562 mg, 2.04 1 mmol, 31%). H NMR (300 MHz, CDCl3) δ: 9.83 (t, J = 2.2 Hz, 1H); 7.85 (dd, J = 5.6, 3.0 Hz, 2H); 7.74 (dd, J = 5.6, 3.0 Hz, 2H); 5.14 (t, J = 4.6 Hz, 1H); 4.67-4.59 (m, 1H); 4.51- 4.44 (m, 2H); 4.07 (dd, J = 10.5, 4.8 Hz, 2H); 2.76 (dd, J = 4.6, 2.3 Hz, 2H).

2-(2-(2-(3-fluoro-6-methoxy-1,5-naphthyridin-4-yl)-2-hydroxyethyl)-1,3-dioxan-5- yl)isoindoline-1,3-dione (33). To a solution of Compound 19 (137 mg, 0.533 mmol, 1.07 equiv relative to compound 32) in THF (6 mL), n-Hexyllithium (2.5M, 0.213 mL, 0.533 mmol, 1.0 equiv relative to compound 19) was dropwised slowly at -78 °C under N2 protection. The reaction mixture lasted 30 min. Then the mixture was dropwised slowly into a solution of compound 32 (137.6 mg, 0.5 mmol, 1.0 equiv.) in THF at -78 °C under

N2 protection. The reaction mixture lasted 2 hours at -78 °C, then the temperature was raised to -20 °C gradually. After 3 hours, the reaction was quenched by the addition of

NH4Cl solution (5 mL). Chromatography was done on silica gel with hexane/ethyl acetate (1:1) to give product as a white solid (40.4 mg, 0.0891 mmol, 18%). 1H NMR (300 MHz,

CDCl3) δ: 8.65 (s, 1H); 8.25 (d, J = 9.2 Hz, 1H); 7.83 (dd, J = 5.5, 3.0 Hz, 2H); 7.73 (dd, J = 5.5, 3.0 Hz, 2H); 7.12 (d, J = 9.1 Hz, 1H); 6.10 (d, J = 10.4 Hz, 1H); 5.68-5.56 (m, 1H); 4.98 (dd, J = 6.5, 3.8 Hz, 1H); 4.62 (m, 1H); 4.45 (t, J = 10.7 Hz, 1H); 4.43 (t, J = 10.7 Hz,

162

1H); 4.15-3.95 (m, 2H); 4.09 (s, 3H); 2.54 (ddd, J = 13.9, 8.8, 3.8 Hz, 1H); 2.22 (t, J = 13.9, 6.4, 5.2 Hz, 1H).

2-(5-amino-1,3-dioxan-2-yl)-1-(3-fluoro-6-methoxy-1,5-naphthyridin-4-yl)ethanol (34). Compound 33 (40.2 mg, 0.0891 mmol, 1.0 equiv.) was suspended in ethyl acetate (2 mL), ethanolamine (0.081 mL, 1.34 mmol, 15.0 equiv.) was added and the mixture was heated to 70 °C. The reaction mixture lasted overnight and then washed by brine. Chromatography was done on silica gel with methanol/DCM (1:20) to give product as 1 yellow oil (18.9 mg, 0.0585 mmol, 66%). H NMR (300 MHz, CDCl3) δ: 8.62 (d, J = 0.9 Hz, 1H); 8.22 (d, J = 9.2 Hz, 1H); 7.09 (d, J = 9.1 Hz, 1H); 5.57 (dd, J = 8.7, 5.0 Hz, 1H); 4.68 (dd, J = 6.6, 3.8 Hz, 1H); 4.16-4.04 (m, 2H); 4.05 (s, 3H); 3.23 (t, J = 10.6 Hz, 1H); 3.21 (t, J = 10.5 Hz, 1H); 3.11-2.97 (m, 1H); 2.47 (ddd, J = 13.8, 8.7, 3.8 Hz, 1H); 2.15 13 (ddd, J = 13.9, 6.5, 5.0 Hz, 1H). C NMR (75 MHz, CDCl3) δ: 162.0, 156.7, 153.3, 141.3, 141.02, 140.95, 138.89, 138.86, 138.5, 130.7, 130.5, 115.75, 115.71, 99.3, 73.6, 73.5, 65.01, 64.96, 54.4, 44.3, 43.0.

Reductive amination general procedure for 35-40: To a solution of amine (0.2 mmol) in methanol (2 mL) was added the requisite aldehyde (0.2 mmol) and zinc chloride (2 mg, 0.015 mmol, 0.07 equiv). The mixture was stirred at room temperature for 30 min, followed by addition of sodium cyanoborohydride (40 mg, 0.6 mmol, 3.0 equiv). The reaction mixture was stirred at room temperature overnight and then purified by chromatography on silica gel with DCM/methanol (30:1). Occasionally, the product obtained from flash chromatography was contaminated by a BH3 adduct, seen in the 1H NMR spectrum as four broad peaks from 0.00-0.88 ppm. In these instances, the column-purified material was dissolved in methanol (2 mL) and stirred overnight at ambient temperature with Amberlite

163

IRA743 free base (ca. 100 mg). The pure title compound was then obtained by removal of the resin by filtration and removal of the solvent under reduced pressure.

2-(5-(((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)amino)-trans-1,3-dioxan-2-yl)-1- (3-fluoro-6-methoxy-1,5-naphthyridin-4-yl)ethan-1-ol (35). The title compound was prepared in 77% yield following the general method and obtained as a white solid. 1H NMR

(300 MHz, CDCl3) δ: 8.62 (s, 1H); 8.22 (d, J=9.1 Hz, 1H); 7.10 (d, J=9.1 Hz, 1H); 6.81- 6.71 (m, 3H); 6.03 (brs, 1H); 5.57 (brs, 1H); 4.70 (dd, J=6.5, 3.8 Hz, 1H); 4.23 (s, 4H); 4.21-4.08 (m, 2H); 4.05 (s, 3H); 3.67 (s, 2H); 3.29 (t, J = 10.6 Hz, 1H); 3.27 (t, J = 10.5 Hz, 1H); 3.02-2.89 (m, 1H); 2.46 (ddd, J=13.9, 8.7, 3.8 Hz, 1H); 2.13 (ddd, J=13.8, 6.4, 13 5.2 Hz, 1H). C NMR (75 MHz, CDCl3) δ: 162.0, 156.7, 153.3, 143.6, 142.9, 141.3, 141.1, 141.0, 138.9, 138.8, 138.5, 133.6, 130.7, 130.5, 121.0, 117.4, 116.9, 115.73, 115.70, 99.6, 71.8, 71.7, 65.03, 64.97, 64.49, 64.46, 54.4, 50.9, 49.7, 43.1. HRMS (ESI) m/z calc’d for + C24H26FN3O6Na [M+Na] : 494.1703; found: 494.1696.

2-(5-(((2,3-dihydro-[1,4]dioxino[2,3-c]pyridin-7-yl)methyl)amino)-trans-1,3-dioxan- 2-yl)-1-(3-fluoro-6-methoxy-1,5-naphthyridin-4-yl)ethan-1-ol (36). The title compound was prepared in 73% yield following the general method and obtained as a white solid. 1H

NMR (300 MHz, CDCl3) δ: 8.62 (d, J=1.0 Hz, 1H); 8.22 (d, J=9.1 Hz, 1H); 8.08 (s, 1H); 7.09 (d, J=9.1 Hz, 1H); 6.76 (s, 1H); 6.05 (brs, 1H); 5.57 (br dd, J=8.4, 4.7 Hz, 1H); 4.71 (dd, J=6.6, 3.8 Hz, 1H); 4.33-4.30 (m, 2H); 4.27-4.24 (m, 2H); 4.23-4.10 (m, 2H); 4.04 (s, 3H); 3.76 (s, 2H); 3.36 (t, J=10.6 Hz, 1H); 3.34 (t, J=10.6 Hz, 1H); 3.00-2.88 (m, 1H); 2.46

164

(ddd, J=13.9, 8.7, 3.8 Hz, 1H); 2.13 (ddd, J=13.9, 6.6, 4.9 Hz, 1H). 13C NMR (75 MHz,

CDCl3) δ: 162.0, 156.7, 153.2, 152.8, 150.4, 141.3, 141.1, 141.0, 140.4, 139.0, 138.90, 138.86, 138.5, 130.7, 130.5, 115.74, 115.71, 110.8, 99.7, 71.6, 71.5, 65.1, 65.02, 64.96, + 64.2, 54.4, 52.0, 50.0, 43.1. HRMS (ESI) m/z calc’d for C23H26FN4O6 [M+H] : 473.1836; found: 473.1838.

6-(((2-(2-(3-fluoro-6-methoxy-1,5-naphthyridin-4-yl)-2-hydroxyethyl)-trans-1,3- dioxan-5-yl)amino)methyl)-2H-pyrido[3,2-b][1,4]thiazin-3(4H)-one (37). The title compound was prepared in 60 % yield following the general method and obtained as a 1 white solid. H NMR (300 MHz, CDCl3) δ: 8.73 (brs, 1H); 8.63 (s, 1H); 8.24 (d, J=9.1 Hz, 1H); 7.57 (d, J=7.8 Hz, 1H); 7.10 (d, J=9.1 Hz, 1H); 6.94 (d, J=7.8 Hz, 1H); 6.07 (brs, 1H); 5.57 (brs, 1H); 4.72 (dd, J=6.4, 3.8 Hz, 1H); 4.18 (m, 2H); 4.05 (s, 3H); 3.82 (s, 2H); 3.46 (s, 2H); 3.37 (t, J=10.6 Hz, 1H); 3.35 (t, J=10.5 Hz, 1H); 3.03-2.86 (m, 1H); 2.47 (ddd, 13 J=13.9, 8.7, 3.6 Hz, 1H); 2.21-2.06 (m, 1H). C NMR (75 MHz, CDCl3) δ: 165.7, 162.0, 156.5, 153.2, 148.4, 141.3, 141.1, 138.9, 138.5, 136.4, 130.6, 130.5, 117.9, 115.8, 114.1, 99.7, 71.5, 71.4, 65.0, 54.5, 51.6, 50.1, 43.1, 29.8. HRMS (ESI) m/z calc’d for + C23H25FN5O5S [M+H] : 502.1560; found: 502.1573.

6-(((2-(2-(3-fluoro-6-methoxy-1,5-naphthyridin-4-yl)-2-hydroxyethyl)-trans-1,3- dioxan-5-yl)amino)methyl)-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one (38). The title compound was prepared in 56 % yield following the general method and obtained as a 1 white solid. H NMR (300 MHz, CDCl3) δ: 8.63 (d, J=0.9 Hz, 1H); 8.24 (brs, 1H); 8.24 (d,

165

J=9.1 Hz, 1H); 7.20 (d, J=8.0 Hz, 1H); 7.11 (d, J=9.2 Hz, 1H); 6.90 (d, J=8.1 Hz, 1H); 6.08 (d, J=10.6 Hz, 1H); 5.58 (m, 1H); 4.73 (dd, J=6.6, 3.8 Hz, 1H); 4.65 (s, 2H); 4.24- 4.12 (m, 2H); 4.06 (s, 3H); 3.79 (s, 2H); 3.36 (t, J=10.6 Hz, 1H); 3.34 (t, J=10.6 Hz, 1H); 3.03-2.87 (m, 1H); 2.48 (ddd, J=13.9, 8.8, 3.8 Hz, 1H); 2.14 (ddd, J=13.9, 6.6, 4.9 Hz, 1H). 13 C NMR (75 MHz, CDCl3) δ: 165.3, 162.1, 153.3, 151.6, 143.7, 141.4, 141.1, 141.0, 140.2, 139.0, 138.6, 138.4, 130.5, 130.4, 124.3, 118.2, 115.8, 99.7, 71.7, 71.6, 67.4, 65.1, 54.5, + 51.6, 50.1, 43.2, 29.9. HRMS (ESI) m/z calc’d for C23H25FN5O6 [M+H] : 486.1789; found: 486.1798.

2-(5-((3,4-dichlorobenzyl)amino)-trans-1,3-dioxan-2-yl)-1-(3-fluoro-6-methoxy-1,5- naphthyridin-4-yl)ethan-1-ol (39). The title compound was prepared in 68 % yield 1 following the general method and obtained as a white solid. H NMR (300 MHz, CDCl3) δ: 8.62 (d, J=0.9 Hz, 1H); 8.22 (d, J=9.2 Hz, 1H); 7.40 (d, J=2.0 Hz, 1H); 7.36 (d, J=8.2 Hz, 1H); 7.12 (dd, J=8.2, 2.0 Hz, 1H); 7.10 (d, J=9.1 Hz, 1H); 6.07 (br d, J=8.7 Hz, 1H); 5.57 (s, 1H); 4.71 (dd, J=6.7, 3.8 Hz, 1H); 4.19 (ddd, J=10.8, 4.7, 2.3, 1H), 4.14 (ddd, J=10.8, 4.7, 2.3, 1H); 4.05 (s, 3H); 3.75 (s, 2H); 3.31 (t, J=10.6 Hz, 1H); 3.28 (t, J=10.6 Hz, 1H); 3.02-2.84 (m, 1H); 2.47 (ddd, J=13.9, 8.8, 3.8 Hz, 1H); 2.13 (ddd, J=13.9, 6.6, 13 4.9 Hz, 1H). C NMR (75 MHz, CDCl3) δ: 162.0, 156.6, 153.2, 141.3, 141.02, 140.95, 140.6, 138.88, 138.86 (overlapping signal), 138.5, 132.7, 131.2, 130.6, 130.5, 130.4, 129.9, 127.3, 115.77, 115.73, 99.7, 71.6, 71.5, 65.01, 64.95, 54.4, 50.2, 50.0, 43.1. HRMS (ESI) + m/z calc’d for C22H23Cl2FN3O4 [M+H] : 482.1050; found: 482.1048.

2-(5-(((6,7-dihydro-[1,4]oxathiino[2,3-c]pyridazin-3-yl)methyl)amino)-trans-1,3-

166 dioxan-2-yl)-1-(3-fluoro-6-methoxy-1,5-naphthyridin-4-yl)ethan-1-ol (40). The title compound was prepared in 41 % yield following the general method and obtained as a 1 white solid. H NMR (300 MHz, CDCl3) δ: 8.63 (d, J=0.9 Hz, 1H); 8.23 (d, J=9.2 Hz, 1H); 7.26 (s, 1H); 7.10 (d, J=9.2 Hz, 1H); 6.06 (br d, J=9.7 Hz, 1H); 5.57 (m, 1H); 4.72 (dd, J=6.6, 3.8 Hz, 1H); 4.69-4.61 (m, 2H); 4.27-4.14 (m, 2H); 4.05 (s, 3H); 3.96 (s, 2H); 3.36 (t, J=10.6 Hz, 1H); 3.34 (t, J=10.6 Hz, 1H); 3.25-3.17 (m, 2H); 3.02-2.90 (m, 1H); 2.47 (ddd, J=13.8, 8.8, 3.8 Hz, 1H); 2.13 (ddd, J=13.9, 6.6, 4.8 Hz, 1H). 13C NMR (75 MHz,

CDCl3) δ: 162.0, 160.1, 159.5, 156.7, 156.3, 153.9, 153.3, 153.2, 141.3, 141.1, 141.0, 138.91, 138.88, 138.5, 130.6, 130.5, 126.0, 125.2, 115.78, 115.74, 99.7, 71.5, 71.4, 66.3,

65.02, 64.97, 54.5, 50.22, 50.19, 43.1, 25.7. HRMS (ESI) m/z calc’d for C22H25FN5O5S [M+H]+: 490.1560; found: 490.1573.

2-(2-(2-(3-fluoro-6-methoxy-1,5-naphthyridin-4-yl)-2-hydroxyethyl)-trans-1,3- dioxan-5-yl)isoindoline-1,3-dione (42). To a solution of commercial 3-fluoro-6- methoxyquinoline 41 (566.4 mg, 3.2 mmol, 1.0 equiv.) in THF (30 mL), lithium diisopropylamide (1.0 M, 3.84 mL, 3.84 mmol, 1.2 equiv) was dropwised slowly at -78 °C under N2 protection. The reaction mixture lasted for 3 hours. Then the mixture was dropwised slowly into a solution of intermediate 32 (1232 mg, 4.47 mmol, 1.4 equiv.) in

THF at -78 °C under N2 protection. The reaction mixture lasted 1 hour at -78 °C, then the reaction was quenched by the addition of NH4Cl solution (6 mL). The organics were extracted by DCM and concentrated as crude products. Chromatography was done on silica gel with methanol/DCM (1:50) to give product as a white solid (150.0 mg, 0.332 mmol, 1 10%). H NMR (300 MHz, CDCl3) δ: 8.59 (d, J = 2.0 Hz, 1H); 8.00 (d, J = 9.2 Hz, 1H); 7.88-7.82 (m, 2H); 7.80 (d, J = 2.7 Hz, 1H); 7.79-7.71 (m, 2H); 7.32 (dd, J = 9.2, 2.7 Hz, 1H); 5.90 (br d, J = 9.6 Hz, 1H); 5.01 (t, J = 4.6 Hz, 1H); 4.69 (m, 1H); 4.49 (t, J = 10.9, 1H); 4.49 (t, J = 11.0, 1H); 4.17-4.06 (m, 2H); 3.96 (s, 3H); 3.32 (brs, 1H); 2.65 (ddd, J =

167

14.5, 9.6, 4.8 Hz, 1H); 2.20-2.11(m, 1H).

2-(5-amino-trans-1,3-dioxan-2-yl)-1-(3-fluoro-6-methoxyquinolin-4-yl)ethan-1-ol (43). Compound 42 (150 mg, 0.33 mmol, 1.0 equiv.) was suspended in ethyl acetate (10 mL), ethanolamine (0.30 mL, 4.98 mmol, 15.0 equiv.) was added and the mixture was heated to 70 °C. The reaction mixture lasted overnight and then washed by brine， extracted by DCM three times. The combined organic layer was concentrated to dryness as crude product (69.3 mg, 0.215mmol), and then used directly in next step.

2-(5-(((6,7-dihydro-[1,4]oxathiino[2,3-c]pyridazin-3-yl)methyl)amino)-trans-1,3- dioxan-2-yl)-1-(3-fluoro-6-methoxyquinolin-4-yl)ethan-1-ol (44). To a solution of compound 43 (69.3 mg, 0.215 mmol, 1.0 equiv.) in methanol (2 mL) was added commercial 6,7-dihydro-[1,4]oxathiino[2,3-c]pyridazine-3-carbaldehyde (41.2 mg, 0.215 mmol, 1.0 equiv.) and zinc chloride (2 mg, 0.015 mmol, 0.07 equiv). The mixture was stirred at room temperature for 30 min, followed by addition of sodium cyanoborohydride (43 mg, 0.645 mmol, 3.0 equiv). The reaction mixture was stirred at room temperature overnight and then purified by chromatography on silica gel with DCM/methanol (30:1). The title compound was obtained as light yellow solid (53.1 mg, 0.109 mmol, 51%). This 1 compound is not 100% pure. H NMR (300 MHz, MeOD-d4) δ: 8.54 (d, J=2.3 Hz, 1H); 7.92 (d, J=9.3 Hz, 1H); 7.84 (d, J=2.7 Hz, 1H); 7.54 (s, 1H); 7.35 (dd, J=9.3, 2.7 Hz, 1H); 5.70 (dd, J=8.0, 6.0 Hz, 1H); 4.67 (m, 2H); 4.59 (dd, J=5.7, 4.6, Hz, 1H); 4.16 (ddd, J = 10.8, 4.7, 2.2, 1H); 4.11 (ddd, J = 10.8, 4.8, 2.2, 1H); 3.93 (s, 3H); 3.88 (s, 2H); 3.40-3.23 (m, 4H) (this peak is partially obscured by the NMR solvent); 2.87-2.73 (m, 1H); 2.44 (ddd, 168

J=13.9, 8.2, 4.5 Hz, 1H); 2.14 (dt, J=13.9, 6.0 Hz, 1H).

8-bromo-7-fluoroquinolin-2(1H)-one (47). To a solution of commercial 2-bromo-3- fluorophenylamine 45 (7.76 g, 40 mmol, 1.0 equiv.) and commercial methyl 3,3- dimethoxypropionate 46 (6.8 mL, 48 mmol, 1.2 equiv.) in THF (80 mL), sodium bis(trimethylsilyl)amide (2 M in THF, 30 mL, 60 mmol, 1.5 equiv.) was dropped slowly at

0 °C under N2 protection. The reaction mixture lasted for 16 hours at room temperature. Then the reaction was quenched by the addition of citric acid (15.48 g, 80 mmol, 2.0 equiv.) aqueous solution (80 mL). The organic layer was extracted by ethyl acetate and concentrated to a brown oil. Then the organics were dissolved in DCM (80 mL), and concen.

H2SO4 (32 mL) was added slowly at 0 °C. The mixture was stirred for 30 min, and then the solvent was removed under vacuum. Ice-water (450 mL) was added, yellow precipate came out and was collected by Buchner funnel filtration. The precipate was washed by water until the PH value of filtrate was 7. Then the yellow solid was rinsed by acetonitrile (10 mL x3). After drying, pure product was obtained as light yellow solid (8.28 g, 34.2 mmol, 1 86%). H NMR (300 MHz, CDCl3) δ: 9.13 (brs, 1H); 7.70 (d, J = 9.6 Hz, 1H); 7.52 (dd, J = 8.7, 5.5 Hz, 1H); 7.03 (t, J = 8.4 Hz, 1H); 6.63 (d, J = 9.6 Hz, 1H).

8-bromo-2-chloro-7-fluoroquinoline (48). To a solution of compound 47 (8.27 g, 34.15 mmol, 1.0 equiv.) in DMF (3.95 mL) and toluene (75 mL), phosphoryl chloride (2.54 mL, 27.24 mmol, 0.80 equiv.) was dropped slowly at 95 °C. The reaction mixture lasted for 30 min at 95 °C. The reaction mixture was cooled down to 0 °C and sodium hydroxide (4.1 g, 102.5 mmol, 3.0 equiv) aqueous solution (30 mL) was added. The mixture was washed by

169 saturated brine and extracted by DCM 3 times. The combined organic layer was concentrated to dryness as crude product. Chromatography was done on silica gel with pure DCM to give target product as a white solid (7.78 g, 29.9 mmol, 88%). 1H NMR (300 MHz,

CDCl3) δ: 8.11 (d, J = 8.5 Hz, 1H); 7.79 (dd, J = 9.0, 5.7 Hz, 1H); 7.43 (d, J = 8.5 Hz, 1H); 7.41 (dd, J = 8.9, 8.1 Hz, 1H).

8-bromo-7-fluoro-2-methoxyquinoline (49). To a solution of compound 48 (7.78 g, 29.9 mmol, 1.0 equiv.) in toluene (80 mL), NaOMe (4845.6 mg, 89.7 mmol, 3.0 equiv.) in methanol (16 mL) solution was dropped at 80 °C. The reaction mixture lasted for 90 min at 80 °C. The reaction mixture was cooled down to 0 °C, and then the PH value of the mixture was adjusted to 7 by addition of HCl solution. The mixture was extracted by DCM. The combined organic layer was concentrated to dryness as crude product. Chromatography was done on silica gel with hexane/ethyl acetate (20:1) to give target 1 product as a white solid (4593.9 mg, 17.94 mmol, 60%). H NMR (300 MHz, CDCl3) δ: 7.96 (d, J = 8.8 Hz, 1H); 7.66 (dd, J = 8.8, 5.9 Hz, 1H); 7.21 (t, J = 8.5 Hz, 1H); 6.91 (d, J = 8.8 Hz, 1H); 4.16 (s, 3H).

2-(2-((E)-2-(7-fluoro-2-methoxyquinolin-8-yl)vinyl)-trans-1,3-dioxan-5- yl)isoindoline-1,3-dione (50). Compound 49 (3783 mg, 14.77 mmol, 1.0 equiv.), intermediate 17 (3798 mg, 14.76 mmol, 1.0 equiv.), palladium acetate (732.5 mg, 2.93 mmol, 0.2 equiv.), triphenylphosphine (1611.5 mg, 5.86 mmol, 0.4 equiv.) and silver carbonate (8196 mg, 29.3 mmol, 2.0 equiv.) were placed in a three-neck flask under N2 170 protection. Anaerobic and anhydrous mesitylene (250 mL) was added. The mixture was heated to 135 °C and lasted overnight. Then the mixture was filtered through celite and rinsed with DCM. The combined solution was concentrated to dryness and further purified by chromatography on silica gel with hexane/ethyl acetate (3:1) to give the title compound as a light yellow solid (906.1 mg, 2.09 mmol, 14% yield). This compound is not 100% 1 pure. H NMR (300 MHz, CDCl3) δ: 7.94 (d, J = 8.8 Hz, 1H); 7.88-7.83 (m, 2H); 7.77- 7.69 (m, 3H); 7.60 (dd, J = 8.9, 5.9 Hz, 1H); 7.16 (dd, J = 10.7, 8.9 Hz, 1H); 7.05 (dd, J = 16.6, 5.2 Hz, 1H); 6.80 (d, J = 8.8 Hz, 1H); 5.38 (d, J = 5.3 Hz, 1H); 4.82-4.69 (m, 1H); 4.65-4.57 (m, 2H); 4.17 (dd, J = 10.4, 4.7 Hz, 2H); 4.13 (s, 3H).

2-((E)-2-(7-fluoro-2-methoxyquinolin-8-yl)vinyl)-trans-1,3-dioxan-5-amine (51). A mixture of compound 50 (906.1 mg, 2.09 mmol, 1.0 equiv), ethanolamine (1886 μL, 31.3 mmol, 15 equiv.) and ethyl acetate (30 mL) was stirred and heated at 70 °C overnight. The solvent was removed, and the residue was dissolved in DCM and washed with brine. The organic layer concentrated, and the crude product was purified by chromatography on silica gel with DCM/methanol (15:1) to give the title compound as a brown oil (482.1 mg, 1.58 1 mmol, 76% yield). This compound is not 100% pure. H NMR (300 MHz, CDCl3) δ: 7.91 (d, J = 8.8 Hz, 1H); 7.67 (d, J = 16.7 Hz, 1H); 7.57 (dd, J = 8.8, 5.9 Hz, 1H); 7.13 (dd, J = 10.8, 8.9 Hz, 1H); 6.99 (dd, J = 16.4, 4.9 Hz, 1H); 6.86 (d, J = 8.8 Hz, 1H); 5.14 (d, J = 4.9 Hz, 1H); 4.26 (dd, J = 11.2, 4.8 Hz, 2H); 4.09 (s, 3H); 3.44 (t, J = 11.0 Hz, 2H); 3.24- + 3.14 (m, 1H). HRMS (ESI) m/z calc’d for C16H18FN2O3 [M+H] : 305.1301; found: 305.1279.

171

tert-butyl (2-((E)-2-(7-fluoro-2-methoxyquinolin-8-yl)vinyl)-trans-1,3-dioxan -5- yl)carbamate (52). To a solution of compound 51 (482.1 mg, 1.58 mmol, 1.0 equiv.) in DCM (10 mL), triethylamine (445 μL, 3.16 mmol, 2.0 equiv.) and di-tert-butyl dicarbonate (517.3 mg, 2.37 mmol, 1.5 equiv.) were added. The mixture was stirred overnight, and then it was purified by chromatography on silica gel with hexane/ethyl acetate (4:1). Impure title compound was obtained as white solid (416.7 mg, 1.03 mmol, 65%) and used in next step.

tert-butyl (2-((1S)-2-(7-fluoro-2-methoxyquinolin-8-yl)-1,2-dihydroxy ethyl)-trans- 1,3-dioxan-5-yl)carbamate (53). Compound 52 (416.7 mg, 1.03 mmol, 1.0 equiv.) was dispensed in the mixture of tert-butanol (7.8 mL), ethyl acetate (1.5 mL) and H2O (7.8 mL), methanesulfonamide (236 mg, 2.06 mmol, 2.0 equiv.) and AD mixture β (3282 mg, 2.06 mmol, 2.0 equiv.) were added. The mixture was stirred overnight, and then it was diluted with H2O (50 mL) and extracted by ethyl acetate 3 times. Chromatography was done on silica gel with DCM/methanol (30:1) to give target product as a white solid (374 mg, 0.85 1 mmol, 83%). This compound is not 100% pure. H NMR (300 MHz, MeOD-d4) δ: 8.14 (d, J=8.9 Hz, 1H); 7.79 (dd, J=8.9, 5.9 Hz, 1H); 7.22 (dd, J=10.0, 9.0 Hz, 1H); 6.93 (d, J=8.9 Hz, 1H); 6.59 (brs, 1H); 5.64 (d, J=5.0 Hz, 1H); 4.44 (d, J=4.8 Hz, 1H); 4.11-3.99 (m, 3H); 4.04 (s, 3H); 3.79-3.65 (m, 1H); 3.33 (t, J=10.9 Hz, 1H); 3.28 (t, J=10.9 Hz, 1H), 1.41 (s,

172

13 9H). C NMR (75 MHz, MeOD-d4) δ: 163.5, 163.3, 160.2, 157.6, 147.3, 147.1, 141.1, 130.3, 130.2, 123.5, 123.4, 121.4, 121.3, 115.0, 114.7, 113.1, 113.0, 101.7, 80.5, 76.3,

70.50, 70.46, 69.91, 69.86, 54.4, 44.4, 43.3, 28.6. HRMS (ESI) m/z calc’d for C21H28FN2O7 [M+H]+: 439.1881; found: 439.1853.

tert-butyl (2-((4S)-5-(7-fluoro-2-methoxyquinolin-8-yl)-2-oxo-trans-1,3- dioxolan-4- yl)-1,3-dioxan-5-yl)carbamate (54). To a solution of compound 53 (374 mg, 0.853 mmol, 1.0 equiv.) in 2-butanone (9 mL), triethylamine (238 μL, 1.71 mmol, 2.0 equiv.) and 1'- carbonyldiimidazole (208 mg, 1.28 mmol, 1.5 equiv.) were added. The mixture was warmed up to 60 °C and stirred overnight. Chromatography was done on silica gel with DCM/methanol (30:1) to give target product as a white solid (300.4 mg, 0.647 mmol, 76%). 1 H NMR (300 MHz, CDCl3) δ: 7.93 (d, J = 8.9 Hz, 1H); 7.71 (dd, J = 8.9, 6.1 Hz, 1H); 7.11 (t, J = 9.3 Hz, 1H); 6.85 (d, J =8.9 Hz, 1H); 6.47 (d, J =5.8 Hz, 1H); 5.05 (dd, J = 5.8, 3.3 Hz, 1H); 4.78 (d, J = 3.3 Hz, 1H); 4.54 (br d, J =5.9 Hz, 1H); 4.29 (ddd, J = 10.7, 5.0, 1.9 Hz, 1H); 4.19 (ddd, J = 10.7, 5.0, 1.8 Hz, 1H); 4.05 (s, 3H); 4.01-3.85 (m, 1H); 3.45 (t, 13 J = 10.8 Hz, 1H); 3.38 (t, J = 10.8 Hz, 1H); 1.41 (s, 9H). C NMR (75 MHz, CDCl3) δ: 163.6, 163.3, 160.2, 155.4, 155.1, 145.9, 145.8, 139.3, 131.5, 131.4, 122.34, 122.32, 116.1, 116.0, 113.5, 113.2, 113.1, 113.0, 98.3, 80.2, 79.08, 79.06, 70.3, 70.2, 69.9, 69.5, 54.1, 43.2, + 28.3. HRMS (ESI) m/z calc’d for C22H26FN2O8 [M+H] : 465.1673; found: 465.1646.

tert-butyl (2-((S)-2-(7-fluoro-2-methoxyquinolin-8-yl)-1-hydroxyethyl)-trans-1,3-

173 dioxan-5-yl)carbamate (55). Compound 54 (300.4 mg, 0.647 mmol, 1.0 equiv.) was dissolved in ethanol (5 mL) and ethyl acetate (0.8 mL), ammonium formate (226 mg, 3.58 mmol, 5.5 equiv.) was added. The mixture was heated to 70 °C, and 5% palladium on calcium carbonate (26 mg, 0.02 equiv.) was added. Then the mixture was cooled down to 40 °C, and 10% palladium on carbon (6.6 mg, 0.01 equiv.) was added. The reaction mixture was stirred at 40 °C overnight. Chromatography was done on silica gel with hexane/ethyl acetate (30:1) to give target product as a colorless oil (138.9 mg, 0.329 mmol, 51%). 1H

NMR (300 MHz, CDCl3) δ: 7.95 (d, J = 8.9 Hz, 1H); 7.58 (dd, J = 8.8, 6.0 Hz, 1H); 7.15 (t, J = 9.0 Hz, 1H); 6.85 (d, J =8.8 Hz, 1H); 4.41 (d, J = 4.4 Hz, 1H); 4.32-4.21(m, 3H); 4.05-3.99 (m, 1H); 4.06 (s, 3H); 3.98-3.84 (m, 1H); 3.52 (ddd, J = 13.9, 3.5, 1.7 Hz, 1H); 3.39 (ddd, J=13.9, 8.3, 1.9, 1H); 3.34 (t, J=10.8, 1H); 3.33 (t, J=10.8, 1H); 1.43 (s, 9H). 13 C NMR (75 MHz, CDCl3) δ: 163.5, 163.0, 160.2, 155.0, 146.8, 146.7, 139.7, 127.5, 127.4, 122.00, 121.98, 120.4, 120.2, 114.2, 113.8, 112.11, 112.07, 102.5, 80.2, 73.0, 70.1, 54.0, + 43.4, 28.4, 26.2. HRMS (ESI) m/z calc’d for C21H28FN2O6 [M+H] : 423.1931; found: 423.1904.

(S)-1-(5-amino-trans-1,3-dioxan-2-yl)-2-(7-fluoro-2-methoxyquinolin-8-yl)ethan-1-ol (56). Compound 55 (138.9 mg, 0.329 mmol, 1.0 equiv.) was dissolved in DCM (5 mL), trifluoroacetic acid (0.66 mL, 8.62 mmol, 26.2 equiv.) was added. The reaction mixture was stirred for 2 hours and then quenched by saturated Na2CO3 solution (10 mL). The organic layer was collected and the aqueous layer was extracted by DCM three times. The combined organic layer was concentrated to dryness as crude product (104.4 mg, 0.323 mmol), and then used directly in next step.

Reductive amination general procedure for 57-61: To a solution of amine (0.1 mmol) in methanol (2 mL) was added the requisite aldehyde (0.1 mmol) and zinc chloride (1 mg, 0.007 mmol, 0.07 equiv). The mixture was stirred at room temperature for 30 min, followed 174 by addition of sodium cyanoborohydride (20 mg, 0.3 mmol, 3.0 equiv). The reaction mixture was stirred at room temperature overnight and then purified by chromatography on silica gel with DCM/methanol (30:1). The pure title compound was then obtained.

(S)-1-(5-(((2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methyl)amino)-trans-1,3-dioxan-2- yl)-2-(7-fluoro-2-methoxyquinolin-8-yl)ethan-1-ol (57). The title compound was prepared in 66 % yield following the general method and obtained as a white solid. 1H

NMR (300 MHz, CDCl3) δ: 7.96 (d, J=8.8 Hz, 1H); 7.58 (dd, J=8.8, 6.0 Hz, 1H); 7.16 (t, J=9.0 Hz, 1H); 6.85 (d, J=8.8 Hz, 1H); 6.83-6.72 (m, 3H); 4.42 (d, J=4.3 Hz, 1H); 4.30- 4.20 (m, 2H); 4.23 (s, 4H); 4.07 (s, 3H); 4.02-3.97 (m, 1H); 3.70 (s, 2H); 3.51 (ddd, J=13.8, 13 3.4, 1.6 Hz, 1H); 3.44-3.27 (m, 3H); 3.09-2.97 (m, 1H). C NMR (75 MHz, CDCl3) δ: 163.5, 163.0, 160.2, 146.8, 146.7, 143.6, 142.9, 139.6, 133.3, 127.5, 127.4, 122.01, 122.00, 121.1, 120.5, 120.3, 117.4, 117.0, 114.1, 113.8, 112.11, 112.07, 102.7, 73.2, 71.61, 71.56,

64.49, 64.46, 54.0, 50.9, 49.8, 26.42, 26.38. HRMS (ESI) m/z calc’d for C25H28FN2O6 [M+H]+: 471.1931; found: 471.1930.

(S)-1-(5-(((2,3-dihydro-[1,4]dioxino[2,3-c]pyridin-7-yl)methyl)amino)-trans-1,3- dioxan-2-yl)-2-(7-fluoro-2-methoxyquinolin-8-yl)ethan-1-ol (58). The title compound was prepared in 61 % yield following the general method and obtained as a light yellow 1 solid. H NMR (300 MHz, CDCl3) δ: 8.09 (s, 1H); 7.95 (d, J=8.9 Hz, 1H); 7.58 (dd, J=8.8, 6.0 Hz, 1H); 7.15 (t, J=9.0 Hz, 1H); 6.85 (d, J=8.8 Hz, 1H); 6.80 (s, 1H); 4.43 (d, J=4.3 Hz, 1H); 4.34-4.22 (m, 6H); 4.06 (s, 3H); 4.04-3.95 (m, 1H); 3.81 (s, 2H); 3.55-3.33 (m, 13 4H); 3.10-2.97 (m, 1H). C NMR (75 MHz, CDCl3) δ: 163.5, 163.0, 160.3, 152.2, 150.5, 175

146.8, 146.7, 140.5, 139.7, 139.0, 127.5, 127.4, 122.02, 122.01, 120.5, 120.3, 114.2, 113.8, 112.1, 111.0, 102.7, 73.2, 71.26, 71.18, 65.1, 64.2, 54.0, 51.8, 50.0, 26.44, 26.40. HRMS + (ESI) m/z calc’d for C24H27FN3O6 [M+H] : 472.1884; found: 472.1851.

6-(((2-((S)-2-(7-fluoro-2-methoxyquinolin-8-yl)-1-hydroxyethyl)-trans-1,3-dioxan-5- yl)amino)methyl)-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one (59). The title compound was prepared in 70 % yield following the general method and obtained as a light yellow 1 solid. H NMR (300 MHz, CDCl3) δ: 9.06 (brs, 1H); 7.95 (d, J=8.9 Hz, 1H); 7.58 (dd, J=8.9, 6.0 Hz, 1H); 7.21 (d, J=8.1 Hz, 1H); 7.15 (t, J=9.0 Hz, 1H); 6.93 (d, J=8.1 Hz, 1H); 6.85 (d, J=8.8 Hz, 1H); 4.64 (s, 2H); 4.44 (d, J=4.3 Hz, 1H); 4.32-4.24 (m, 2H); 4.06 (s, 3H); 4.04-3.97 (m, 1H); 3.83 (s, 2H); 3.51 (ddd, J=13.8, 3.6, 1.6 Hz, 1H). 3.46-3.32 (m, 13 3H); 3.10-2.97 (m, 1H); C NMR (75 MHz, CDCl3) δ: 165.6, 163.5, 163.0, 160.3, 151.4, 146.8, 146.7, 140.5, 139.7, 138.5, 127.5, 127.4, 124.4, 122.03, 122.01, 120.4, 120.2, 118.3, 114.1, 113.8, 112.14, 112.10, 102.7, 73.1, 71.42, 71.35, 67.4, 54.0, 51.3, 50.1, 26.49, 26.44. + HRMS (ESI) m/z calc’d for C24H26FN4O6 [M+H] : 485.1836; found: 485.1803.

(S)-1-(5-((3,4-dichlorobenzyl)amino)-trans-1,3-dioxan-2-yl)-2-(7-fluoro-2- methoxyquinolin-8-yl)ethan-1-ol (60). The title compound was prepared in 59 % yield 1 following the general method and obtained as a white solid. H NMR (300 MHz, CDCl3) δ: 7.97 (d, J=8.9 Hz, 1H); 7.59 (dd, J=8.8, 6.0 Hz, 1H); 7.44 (d, J=1.6 Hz, 1H); 7.39 (d, J=8.2 Hz, 1H); 7.21-7.11 (m, 2H); 6.86 (d, J=8.8 Hz, 1H); 4.67 (s, 1H); 4.43 (d, J=4.3 Hz, 1H); 4.31-4.23 (m, 2H); 4.07 (s, 3H); 4.04-3.97 (m, 1H); 3.78 (s, 2H); 3.56-3.47 (m, 1H). 13 3.45-3.28 (m, 3H); 3.08-2.95 (m, 1H); C NMR (75 MHz, CDCl3) δ: 163.5, 163.1, 160.3,

176

146.9, 146.7, 139.7, 132.7, 131.4, 130.6, 130.0, 127.6, 127.4, 122.05, 122.03, 120.4, 120.2, 114.2, 113.8, 112.18, 112.14, 102.7, 73.2, 71.5, 54.0, 50.2, 50.1, 26.42, 26.37. HRMS (ESI) + m/z calc’d for C23H24Cl2FN2O4 [M+H] : 481.1097; found: 481.1064.

(S)-1-(5-(((6,7-dihydro-[1,4]oxathiino[2,3-c]pyridazin-3-yl)methyl)amino)-trans-1,3- dioxan-2-yl)-2-(7-fluoro-2-methoxyquinolin-8-yl)ethan-1-ol (61). The title compound was prepared in 72 % yield following the general method and obtained as a light yellow 1 solid. H NMR (300 MHz, CDCl3) δ: 7.96 (d, J=8.9 Hz, 1H); 7.58 (dd, J=8.8, 6.0 Hz, 1H); 7.29 (s, 1H); 7.16 (t, J=9.0 Hz, 1H); 6.85 (d, J=8.8 Hz, 1H); 4.68-4.61 (m, 2H); 4.44 (d, J=4.3 Hz, 1H); 4.36-4.25 (m, 2H); 4.06 (s, 3H); 4.04-3.94 (m, 1H); 3.99 (s, 2H); 3.51 (ddd, J=13.9, 3.5, 1.6 Hz, 1H). 3.44-3.33 (m, 3H); 3.24-3.17 (m, 2H); 3.11-2.98 (m, 1H); 13C

NMR (75 MHz, CDCl3) δ: 163.5, 163.0, 160.2, 160.1, 156.2, 146.8, 146.7, 139.7, 127.5, 127.4, 126.0, 125.3, 122.02, 122.01, 120.4, 120.2, 114.2, 113.8, 112.14, 112.10, 102.7, 73.2, 71.4, 71.3, 66.3, 54.0, 50.23, 50.16, 26.38, 26.33, 25.7. HRMS (ESI) m/z calc’d for + C23H26FN4O5S [M+H] : 489.1608; found: 489.1575.

3,3-diethoxypropanal (63). To a solution of ethyl 3,3-diethoxypropionate (972.6 μL, 5.00 mmol, 1.0 equiv.) in diethyl ether (5 mL) was drop slowly with DIBAL-H solution (1M in Hexane, 5.5 mL, 5.5 mmol, 1.1 equiv.) at -78 °C. The reaction mixture was stirred at -78 °C for 1 hour, and then MeOH (300 μL) was added for quenching. After 15 minutes, saturated aqueous solution of Roche salts (30 mL) was added. The mixture was stirred for another 2 hours and then extracted by diethyl ether twice. The combined organic layer was filtered through celite to filter insoluble impurities out. The organic layer was then concentrated under vacuum gently to give the crude title compound as a colorless oil (550 mg), used

177 without further purification. [37]

3,3-diethoxy-1-(7-fluoro-2-methoxyquinolin-8-yl)propan-1-ol (64). To a solution of Compound 49 (2561 mg, 10.0 mmol, 1.0 equiv.) in THF (50 mL), n-Hexyllithium (2.5M in Hexane, 4 mL, 10.0 mmol, 1.0 equiv.) was dropped slowly at -78 °C under N2 protection. The reaction mixture lasted 30 min at -78 °C. The solution of aldehyde 63 (1662 mg, 12.6 mmol, 1.26 equiv.) in THF (12 mL) was dropped slowly into the reaction mixture. The reaction mixture lasted 2 hours at -78 °C, and then 30 minutes at room temperature. The reaction was quenched by the addition of NH4Cl solution (60 mL). The organic layer was collected and concentrated as crude oil product. Chromatography purification was done on silica gel with hexane/ethyl acetate (3:1) to give product as a white solid (2395 mg, 7.41 1 mmol, 74%). H NMR (300 MHz, CDCl3) δ: 7.99 (d, J = 8.9 Hz, 1H); 7.61 (dd, J = 8.9, 5.9 Hz, 1H); 7.14 (t, J = 9.1 Hz, 1H); 6.89 (d, J = 8.9 Hz, 1H); 5.53 (dd, J = 8.5, 5.3 Hz, 1H); 4.81 (dd, J = 7.1, 4.4 Hz, 1H); 4.05 (s, 3H); 3.71-3.60 (m, 2H); 3.59-3.46 (m, 2H); 2.50 (ddd, J = 13.7, 8.6, 4.4 Hz, 1H); 2.22 (ddd, J = 13.7, 7.1, 5.3 Hz, 1H); 1.19 (td, J = 7.0, 0.8 Hz, 6H).

3-ethoxy-10-fluoro-1-hydroxy-2,3-dihydro-1H,5H-pyrido[3,2,1-ij]quinolin-5-one (67). To a mixture of compound 64 (323.4 mg, 1.0 mmol, 1.0 equiv.) and diol 3 (221 mg, 1.0 mmol, 1.0 equiv.) in chloroform (6 mL), boron trifluoride etherate (254 μL, 2.0 mmol, 2.0 equiv.) was added under N2 protection. The reaction mixture was warmed to reflux and lasted 1 hour. The mixture was cooled down to room temperature, and then quenched by

178 the addition of aqueous NaOH solution (1M, 5 mL). The organic layer was collected and concentrated as crude oil product. Chromatography purification was done on silica gel with hexane/ethyl acetate (3:1) to give product as a white solid (161 mg, 0.62 mmol, 62%). This compound is not 100% pure and appears to be a single diastereomer. 1H NMR (300 MHz,

CDCl3) δ: 7.62 (d, J = 9.6 Hz, 1H); 7.44 (dd, J = 8.6, 6.1 Hz, 1H); 6.97 (dd, J = 10.8, 8.7 Hz, 1H); 6.59 (d, J = 9.5 Hz, 1H); 6.50 (t, J = 2.8 Hz, 1H); 5.53 (dd, J = 11.2, 6.8 Hz, 1H); 3.69 (dq, J = 9.6, 7.0 Hz, 1H); 3.51 (dq, J = 9.6, 7.0 Hz, 1H); 3.01 (brs, 1H); 2.76 (ddd, J = 13.4, 6.7, 3.4 Hz, 1H); 1.99 (ddd, J = 13.5, 11.3, 2.4 Hz, 1H); 1.10 (t, J = 7.0 Hz, 3H).

3.5.2 Biological evaluation assays

Determination of Minimum Inhibitory Concentrations (MICs). MICs against the strains listed in Tables 1, were determined by broth microdilution according to the guidelines established by the Clinical and Laboratory Standards Institute [25].

Topoisomerase Inhibition. The assays were conducted at Inspiralis, Ltd. (Norwich, UK) unless otherwise noted. Average (mean) values are reported in cases where compounds were assayed multiple times. In all experiments, the activity of the enzymes was determined prior to the testing of the compounds and 1 unit (U) was defined as the amount of enzyme required to just fully supercoil or decatenate the substrate. This amount of enzyme was initially used in determination of control inhibitor activity. These experiments were performed in duplicate.

Stocks (10 mM in 100% DMSO) of the compounds were serially diluted to 10% DMSO at concentrations ranging from 50 nM to 1 mM. Drug dilutions or DMSO (3.0 μL) were added to a final reaction mixture of 30 μL where final DMSO concentration was 1% in all. Bands were visualized by ethidium staining for 20 min and destaining for 20 min. Gels were scanned using documentation equipment (GeneGenius, Syngene, Cambridge, UK) and % inhibition levels (where appropriate) were obtained with gel scanning software. (GeneTools, Syngene, Cambridge,UK)

Staphylococcus aureus gyrase supercoiling assay. 1 U of DNA gyrase was incubated with 0.5 μg of relaxed pBR322 DNA in a 30 μL reaction at 37C for 30 min under the following 179 conditions: 40 mM HEPES. KOH (pH 7.6), 10 mM magnesium acetate, 10 mM DTT, 2 mM ATP, 500 mM potassium glutamate and 0.05 mg/mL BSA. Each reaction was stopped by the addition of 30 μL chloroform/iso-amyl alcohol (26:1) and 30 μL Stop Dye (40% sucrose (w/v), 100 mM Tris.HCl (pH 7.5), 10 mM EDTA, 0.5 μg/mL bromophenol blue), before being loaded on a 1.0% TAE gel run at 70V for 2 hr.

Staphylococcus aureus topoisomerase IV decatenation assay. Decatenation assays were performed as per protocol by Inspiralis, Ltd. (Norwich, UK) where indicated (Method A). Briefly, decatenation of 200 ng kDNA was performed at 37C for 30 min in a total reaction volume of 30 µL containing 50 mM Tris.HCl (7.5), 5 mM MgCl2, 5 mM DTT, 1.5 mM ATP, 350 mM potassium glutamate, 0.05 mg/ml BSA, 1% DMSO vehicle control (or compound solution) and 1 unit of topoisomerase IV (defined as that amount of enzyme to just completely decatenate kDNA). Each reaction was stopped by the addition of 30 μL chloroform/isoamyl alcohol (26:1) and 30 μL of a buffer containing 40% (w/v) sucrose, 100 mM Tris-HCl (pH 8), 10 mM EDTA, 0.5 mg/mL bromophenol blue. Aqueous fractions (20 µL) were loaded onto a 1% Agarose gel and run at 70V for 2 hr, stained with ethidium bromide for subsequent UV visualization and quantitation of the percent decatenation in the presence or absence of various concentrations of test compounds, and assessment of 50% inhibitory concentrations. Where noted, results were obtained at The Ohio State University based on slight modifications (Method B) where reactions contained 100 ng kDNA, were incubated for 20 min, and contained a final DMSO concentration of 1.7% in reaction mixtures.

Human topoisomerase II decatenation assay. 1 U of human topo II was incubated with 200 ng kDNA in a 30 μl reaction at 37°C for 30 minutes under the following conditions: 50 mM Tris HCl (pH 7.5), 125 mM NaCl, 10 mM MgCl2, 5 mM DTT, 0.5 mM EDTA, 0.1 mg/ml bovine serum albumin (BSA) and 1 mM ATP. Each reaction was stopped by the addition of 30 μl chloroform/iso-amyl alcohol (26:1) and 30 μl Stop Dye, before being loaded on a 1.0 % TAE gel run at 90V for 2 hours.

Frequency of Spontaneous Resistance Studies. In order to determine the spontaneous mutation frequency of investigational agents, the MIC was first determined using the agar 180 dilution method as recommended by the Clinical and Laboratory Standards Institute [25, 26]. The test agents and ciprofloxacin were assayed using a drug concentration range of 8- 0.008 μg/mL. Triplicate independent inocula of S. aureus ATCC 29213 were evaluated.

Serial dilutions of the experimental compounds were made in DMSO; ciprofloxacin was made in water. Agar plates were prepared by mixing 1.25 mL of concentrated drug (40X the agar dilution MIC) with 48.75 mL of molten (52°C) Mueller-Hinton Agar and pouring into 15-by-150 mm petri dishes, followed by allowing the plates to solidify and dry at room temperature prior to inoculation. Duplicate plates were prepared to contain the agents at 4X and 8X the agar dilution MIC. In addition, a single 4X MIC and 8X MIC plate (standard small petri plates) was prepared and inoculated according to CLSI [25] for the agar dilution assay. This was to confirm that the drug content of the plates was above the MIC, validating that the stock solution of drug(s) was made properly.

The inoculum for the assay was prepared by streaking S. aureus ATCC 29213 onto 6 TSAB plates and growing for approximately 20 hr at 35°C. Using a sterile swab, several well- isolated colonies were removed and resuspended in sterile saline at a heavy concentration equivalent to 1.6 on the turbidimeter. This suspension was diluted 1:10 in order to achieve a target inoculum of 109 CFU on each spontaneous mutation plate. Each plate was inoculated with 0.2 mL of the cell suspension, resulting in each drug being tested at 4X and 8X the MIC in duplicate. In addition, a portion of the inoculum was enumerated by making dilutions and spreading onto TSA, followed by counting colonies after a 24 hr incubation at 35°C.

Once the inoculum was absorbed into the agar, the drug-containing plates were incubated at 35°C for 48 hr and the colonies were counted. The spontaneous mutation frequency was determined by dividing the number of colonies that appeared at a given drug concentration, averaging the counts from the duplicate plates, and dividing by the number of bacteria applied to the agar surface. hERG Inhibition. Assays were conducted at Charles River (Cleveland, OH) using IonWorksTM Barracuda systems (Molecular Devices Corporation, Union City, CA). Evaluations were conducted using four replicates per concentration for each compound. 181

Cisapride was employed as the positive control.

Growth Inhibition. Log-phase parental K562 and cloned K/VP.5 cells were adjusted to 1 x 105 cell/ml and 1.25 x 105 cell/mL respectively, and incubated for 48 hr with 0-200 µM NBTIs after which cells were counted on a model ZBF Coulter counter (Beckman Coulter, Danvers, MA). Growth beyond the starting concentrations in drug-treated versus control cells was ultimately expressed as percent inhibition of control growth. The 50% growth- inhibitory concentration for each NBTI in each cell line was calculated from concentration- response curves generated by use of Sigmaplot 13 (Systat Software, Inc; San Jose, CA). All NBTI were dissolved in 100% DMSO and added to cell suspensions to achieve a final solvent concentration of 0.5%.

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Chapter 3 1. Mayer C, Janin YL. Non-quinolone inhibitors of bacterial type IIA topoisomerases: a feat of bioisosterism. Chem Rev 2014, 114, 2313-2342. 2. Ehmann DE, Lahiri SD. Novel compounds targeting bacterial DNA topoisomerase/DNA gyrase. Curr Opin Pharmacol 2014, 18, 76-83. 3. Black MT, Coleman K. New inhibitors of bacterial topoisomerase GyrA/ParC subunits. Curr Opin Invest Drugs 2009, 10, 804-810. 4. Reck F, Ehmann DE, Dougherty TJ, Newman JV, et al. Optimization of 198

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Appendix. Copies of 1H NMR, 13C NMR and MS of Desired Compounds

The copies of H1 NMR, C13 NMR and Mass Spectrum for desired compounds can be found in the supplementary file of this dissertation.

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