Cost-Effective Synthesis, Bioactivity and Cellular Uptake Study of Aminoglycosides with Antimicrobial and Connexin Hemichannel Inhibitory Activity

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Yagya P. Subedi Utah State University

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This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@USU. It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. COST-EFFECTIVE SYNTHESIS, BIOACTIVITY AND CELLULAR UPTAKE

STUDY OF AMINOGLYCOSIDES WITH ANTIMICROBIAL AND

CONNEXIN HEMICHANNEL INHIBITORY ACTIVITY

Yagya P. Subedi

A dissertation submitted in partial fulfillment of the requirements for the degree

DOCTOR OF PHILOSOPHY

Chemistry

Approved:

______Cheng-Wei Tom Chang, Ph.D. Alvan C. Hengge, Ph.D. Major Professor Committee Member

______Joan M. Hevel, Ph.D. Liaohai Chen, Ph.D. Committee Member Committee Member

______David W. Britt, Ph.D. Richard S. Inouye, Ph.D. Committee Member Vice Provost for Graduate Studies

UTAH STATE UNIVERSITY Logan, Utah

2019 ii

ABSTRACT

Cost-effective synthesis, bioactivity and cellular uptake study of aminoglycosides

with antimicrobial and connexin hemichannel inhibitory activity

Yagya P. Subedi, Doctor of Philosophy in Chemistry

Utah State University, 2019

Major Professor: Dr. Cheng-Wei Tom Chang Department: Chemistry and Biochemistry

More than a billion people suffer from fungal diseases, and these diseases cost billions of dollars worldwide each year. The emergence of a fungal strain resistance to the first-line drugs, azoles, makes the situation even worst. Amphiphilic kanamycin is a new class of compounds with wide-spectrum antifungal activity towards susceptible and azole resistance strains. The higher cost of production and instability at acidic or basic conditions were the limiting factors of these antifungal compounds. Libraries of new 6ʺ modified amphiphilic kanamycins were synthesized with nitrogen and sulfur as the connector atoms. The leads identified after the SAR study of the new compounds have excellent activity towards plant and fungal pathogens and can tolerate different pH conditions. The cost of production of the lead compounds is higher amid improved antifungal activity. A library 6ʹ modified of amphiphilic kanamycin was synthesized from the one-step modification of kanamycin to resolve the cost issue. The lead from the library of 6ʹ amphiphilic kanamycin has similar activity to the lead from 6ʺ compounds. iv

The newly identified lead from the library of 6ʹ modified kanamycin can be synthesized

on a large scale without a need for conventional silica gel column chromatography, and

the cost of production is competitive to currently marketed antifungals.

Newly synthesized amphiphilic aminoglycosides have fungal selective activity.

Using the fluorescent analogs, the fluorescent imaging of fungi, bacteria, and human cells

shows the discrimination in cellular uptake was the reason behind selectivity. These

fluorescent aminoglycosides reported here, with a fluorophore at 6ʹ and 6ʺ, can be used to stain fungi selectively. So these fluorescent analogs also have the potential to be fungal detecting probes.

Abnormal opening of Cx43 hemichannel is associated with various diseases such as neurodegenerative disease, heart diseases, etc. So the Cx43 selective inhibitor may be useful in the treatment of these diseases. In search of an inhibitor selective towards Cx43 hemichannel, newly synthesized 6ʹ and 6ʺ modified kanamycin with aryl substitution and some known compounds were screened for connexin hemichannels inhibitory activity.

The lead identified from these compounds has selective inhibitory activity towards Cx43 hemichannel.

(261 pages)

PUBLIC ABSTRACT

Cost-effective synthesis, bioactivity and cellular uptake study of aminoglycosides with

antimicrobial and connexin hemichannel inhibitory activity

Yagya P. Subedi

Amphiphilic kanamycin is one of the promising class of compounds for the

treatment of fungal infections in plants and animal. Factor that lead to the restricting of compounds for commercialization includes, the higher cost of production and poor stability of the compound. However, the new lead, identified from the synthesis and biological testing, can be synthesized on a large scale with a cost comparable to

commercial antifungals. The newly reported lead is stable at the acidic and basic

conditions. Additionally, this compound has an excellent activity towards Candida

auris, a multidrug-resistant superbug.

Heart disease is the leading cause of death in the United States most of which are

caused by cardiac ischemia and arrhythmias. Abnormal opening of Cx43 hemichannel

can damage the heart muscles and lead to these conditions. A compound which can

selectively inhibit the opening of Cx43 hemichannel may pave the way to reducing the

mortality rate of heart disease. A selective inhibitor towards Cx43 hemichannel is

explored from the synthesis and biological testing of kanamycin derivatives. The

synthesis of the new inhibitor is scalable and cost-effective. vi

ACKNOWLEDGMENTS

First and foremost, I would like to express my appreciations and thanks to my advisor Dr. Cheng-Wei Tom Chang; it was a great pleasure to be his graduate student.

His guidance, motivation, and supports in the last four years transform me from a fluster researcher to a synthetic chemist. His kindness and humbleness are irreplaceable. I also would like to thank Dr. Liaohai Chen for allowing me to work under his supervision in the first two years of PhD and providing constructive comments as a committee member.

I learned biology and analytical techniques while working in Dr. Chen’s lab. I am thankful to other committee members Dr. Alvan C. Hengee, Dr. Joan M. Hevel, and Dr.

David W. Britt, for encouragement and guidance throughout the journey to doctorate. I heartily appreciate the invaluable comments and suggestions from all of you. A special thanks to Dr. Hengge for helping to establish a human cell culture lab from the departmental support. It would be hard to complete all of my projects without this lab.

I am grateful to our collaborator Dr. Jon Y. Takemoto from the Biology department at Utah State University for teaching me the biology aspect of my antifungal projects and his generosity. I never get a feel Takemoto lab is not my primary research lab while using resources from his lab. Thank you, Dr. Takemoto, for letting me use all the lab resources. I am also grateful to Dr. Michelle Grilley from the Takemoto lab for helping in the antifungal assay, teaching biological assays and helping me with the grammatical correction in this dissertation. I also would like to thank Dr. Guillermo A.

Altenberg and his lab members for performing the connexin inhibitory assay of the compounds.

I am thankful to the undergraduate researchers Paul Roberts, Heath Montgomery, vii

Joey Rapp, Bjorn Rodriguez, Noah Thackeray, Gavin Nichols, Jeffrey Wight, Xinrui

Peng and summer interns David Kennedy, Aleksei Ananin, Greg Becker for their helping hand in my research. It was a wonderful experience working with you all. I am also thankful to graduate students of the lab, Dr. Jaya P. Shrestha, Dr. Madher N. Alfindee,

Uddav Pandey, for maintaining a friendly environment in the lab and helping in my graduate work.

I would like to thank Cindy Weatbrook, and Margaret Dobrowolska for their help in all the difficulties and providing an exciting environment in the chemistry and biochemistry department. Both of you, marvelous lady, will be in my memory forever. I am thankful to Chemistry and Biochemistry, Utah State University, for the opportunity to pursue PhD degree.

I am indebted to my parents for continuous support, love, care, and seeing their success in my progress. Thank you, Buba and Mummy, for inspiration to see a big dream and teaching to achieve that through hard work. I am also grateful to my sister and brother for their love and care throughout student life. Finally, I would like to thank all the friends and relatives who have been around physically or emotionally.

Yagya P. Subedi, 2019

viii

CONTENTS

Page

ABSTRACT ……………………………………………………………………………. iii

PUBLIC ABSTRACT …………………………………………………………………... v

ACKNOWLEDGMENTS ………………………………….…………………………... vi

LIST OF TABLES ………………………………………..………………………...…... ix

LIST OF FIGURES ……………………………………..………………………………. x

LIST OF SCHEMES …………………………………..……………………………... xvii

LIST OF ABBREVIATIONS ………………………..……………………………… xviii

CHAPTER

1. GENERAL INTRODUCTION ………………………………………………...... 1

2. SCALABLE AND COST-EFFECTIVE TOSYLATION-MEDIATED

SYNTHESIS OF ANTIFUNGAL AND FUNGAL DIAGNOSTIC

6″-MODIFIED AMPHIPHILIC ANAMYCINS ……………………………….... 34

3. DEVELOPMENT OF FUNGAL SELECTIVE AMPHIPHILIC KANAMYCIN:

COST-EFFECTIVE SYNTHESIS AND USE OF FLUORESCENT

ANALOGS FOR MODE OF ACTION INVESTIGATION ……………………. 64

4. AMPHIPHILIC AMINOGLYCOSIDES AS SELECTIVE INHIBITORS OF

CX43 CONNEXIN HEMICHANNEL …………………………………………. 103

5. SUMMARY AND CONCLUSION ………………………….………………… 133

APPENDICES ……………………………………………………….……………..… 136

CURRICULUM VITAE ……………………………………………..……………….. 237 ix

LIST OF TABLES

Table Page

2-1. Antifungal activity of AK ………………………………………………….……… 41

2-2. MICs of AK against bacterial strains ……………………………………….….…. 43

2-3. IC50 value of the AK against HeLa cells …………………………………….….… 44

3-1. MICs of AKs against fungi ...... 71

3-2. Minimum inhibitory concentration of AKs against Candida sp. ………………..... 72

3-3. MICs of 2f and 2g against human fungal pathogens ...... 73

3-4. MICs of AKs against bacterial strains …………………………………………….. 75

4-1. IC50 values of commercial aminoglycosides towards Cx26 and Cx43…………... 106

4-2. IC50 values of 4″-6″ benzyl substituted kanamycin towards Cx26

and Cx43 ……………………………………………………………………….… 107

4-3. Percentage of inhibition of Cx26 and Cx43 dependent growth complementation

of 6″ modified kanamycin ………………………………………...... 112

4-4. Percentage of inhibition of Cx26 and Cx43 dependent growth complementation

of 6ʹ modified kanamycin …………………………………………..…...………. 113

4-5. IC50 values of 6″ and 6ʹ modified kanamycin towards Cx26 and

Cx43 HCs ……………………………………………………………….……… 115

B-1. IC50 values of the compounds …………...………………………………………. 177

LIST OF FIGURES

Figure Page

1-1. Structure of streptomycin, 2-deoxystreptamine, and kanamycin and

neomycin class aminoglycosides …………………………………………...... 2

1-2. Aminoglycosides modifying enzymes (AMEs) for the modification of

different groups of kanamycin A and neomycin B ………………………...... 3

1-3. Structure of antibacterial amphiphilic aminoglycosides ………………………….... 5

1-4. Structure of FG compounds. [A] FG08, [B] FG compounds with their

minimum inhibitory concentrations ………………………………………………. 7

1-5. Schematic representation of Connexin monomer (top), hemichannel (HC),

and gap junction channel (GJC)…………………………………………………. 14

2-1. Structure of kanamycin class aminoglycosides …………………………...... 37

2-2. Cytotoxicity of AK against HeLa cells ……………………………………………. 45

2-3. Images of fungi and bacteria incubated with 7 ……………………………………. 47

3-1. Structures of selected kanamycin class antibiotics and AKs ...... 67

3-2. Kinetic membrane permeabilization study of the AKs ...... 77

3-3. Images of fungi treated with fluorescent 2j ...... 79

3-4. Images of fungi treated with fluorescent 2i ...... 80

3-5. Kinetic membrane permeabilization study of 2i and 2j with PI ...... 82

3-6. Cellular ROS production ...... 84

4-1. Structure of previously reported amphiphilic kanamycin …………………..…… 110

4-2. Percentage inhibition of Cx43 HC by 6″ and 6ʹ modified AKs …...…………….. 117

4-3. Cytotoxicity of lead AKs …...……………………………………………………. 118 xi

A-1. Excitation and emission fluorescent spectra of compound 7 in water ……...…… 138

A-2. 1H NMR of 6''-O-Toluenesulfonyl-1,3,6',3''-tetra-N-

(tert-butoxycarbonyl)kanamycin A (2) in CD3OD ………………………….…… 139

A-3. 13C NMR of 6''-O-Toluenesulfonyl-1,3,6',3''-tetra-N-

(tert-butoxycarbonyl)kanamycin A (2) in CD3OD …………………………..…. 140

1 A-4. H NMR of 6″-O-Toluenesulfonylkanamycin A (3) in D2O ……………………. 141

13 A-5. C NMR of 6″-O-Toluenesulfonylkanamycin A (3) in D2O ………………….... 142

1 A-6. H NMR of 6″-(1-Hexylamino)-6″-deoxykanamycin A (4a) in D2O ………….... 143

13 A-7. C NMR of 6″-(1-Hexylamino)-6″-deoxykanamycin A (4a) in D2O …………... 144

1 A-8. H NMR of 6″-(1-Octylamino)-6”-deoxykanamycin A (4b) in D2O ………….... 145

A-9. 13C NMR of 6″-(1-Octylamino)-6”-deoxykanamycin A (4b)

in D2O …………………………………………………………………………... 146

A-10. 1H NMR of 6″-(1-Decylamino)-6″-deoxykanamycin A (4c)

in D2O ………………………………………………………………..………… 147

A-11. 13C NMR of 6″-(1-Decylamino)-6″-deoxykanamycin A (4c)

in D2O ……………………………………………………………………...…… 148

A-12. 1H NMR of 6″-(1-Dodecylamino)-6”-deoxykanamycin A (4d)

in D2O …………………………………………………………………………... 149

A-13. 13C NMR of 6″-(1-Dodecylamino)-6”-deoxykanamycin A (4d)

in D2O …………………………………………………………………………... 150

A-14. 1H NMR of 6″-(1-Tetradecylamino)-6”-deoxykanamycin A (4e)

in D2O ……………………………………………………………………..…… 151

A-15. 13C NMR of 6″-(1-Tetradecylamino)-6”-deoxykanamycin A (4e) xii

in D2O ………………………………………………………………………..…. 152

A-16. 1H NMR of 6″-(1-Hexadecylamino)-6”-deoxykanamycin A (4f)

in CD3OD ………………………………………………………………………. 153

A-17. 13C NMR of 6″-(1-Hexadecylamino)-6”-deoxykanamycin A (4f)

in CD3OD ………………………………………………………………………. 154

A-18. 1H NMR of 6″-(1-Hexylmercapto)-6″-deoxykanamycin A (5a)

in D2O ………………………………………………………………………….. 155

A-19. 13C NMR of 6″-(1-Hexylmercapto)-6″-deoxykanamycin A (5a)

in D2O ………………………………………………………………….………. 156

A-20. 1H NMR of 6″-(1-Octylmercapto)-6”-deoxykanamycin A (5b)

in D2O ………………………………………………………………………….. 157

A-21. 13C NMR of 6″-(1-Octylmercapto)-6”-deoxykanamycin A (5b)

in D2O ………………………………………………………………………….. 158

A-22. 1H NMR of 6″-(1-Decylmercapto)-6”-deoxykanamycin A (5c)

in D2O ………………………………………………………………………….. 159

A-23. 13C NMR of 6″-(1-Decylmercapto)-6”-deoxykanamycin A (5c)

in D2O ………………………………………………………………………….. 160

A-24. 1H NMR of 6″-(1-Dodecylmercapto)-6”-deoxykanamycin A (5d)

in D2O ………………………………………………………………..………… 161

A-25. 13C NMR of 6″-(1-Dodecylmercapto)-6”-deoxykanamycin A (5d)

in D2O ………………………………………………………………….…….… 162

A-26. 1H NMR of 6″-(1-Tetradecylmercapto)-6”-deoxykanamycin A (5e)

in CD3OD ……………………………………………………………………... 163 xiii

A-27. 13C NMR of 6″-(1-Tetradecylmercapto)-6”-deoxykanamycin A (5e)

in CD3OD ……………………………………………………………………... 164

A-28. 1H NMR of 6″-(1-Hexadecylmercapto)-6”-deoxykanamycin A (5f)

in CD3OD ……………………………………………………………………… 165

A-29. 13C NMR of 6″-(1-Hexadecylmercapto)-6”-deoxykanamycin A (5f)

in CD3OD …………………………………………………………………….... 166

A-30. 1H NMR of 6″-(2-(6-hydroxy-3-oxo-3H-xanthen-9-yl))benzamido)-6″-

deoxykanamycin A (7) in D2O ……………………………………………..…. 167

A-31. 13C NMR of 6″-(2-(6-hydroxy-3-oxo-3H-xanthen-9-yl))benzamido)-6″-

deoxykanamycin A (7) in D2O ………………………………………………… 168

1 1 A-32. H- H COSY of 6''-O-Toluenesulfonylkanamycin A (3) in D2O …………...…. 169

A-33. 1H-1H COSY of 6''-O-Toluenesulfonylkanamycin A (3)

(zoomed version) in D2O ………………………………………………………. 170

B-1. Excitation and emission fluorescent spectra of 2i [A] and 2j [B]

in water ………………………………………………………………………….. 172

B-2. Image of Candida albicans 64124 treated with PI after 2 hours

incubation ………………………………………………………………………. 173

B-3. Images of mammalian cells treated with 2j. Hela cells incubated with 2j

at 32 µg/mL for 5 min. (A and B) and 60 min. (C and D) ……...………………. 173

B-4. Images of bacteria treated with 2j ………………………………………..……… 175

B-5. Cell viability of the HeLa cells at different concentrations of the

compounds ……………………………………………………………………….. 177

B-6. 1H-NMR of 2,5-dioxopyrrolidin-1-yl 2-phenylquinoline-4-carboxylate xiv

(1h) in CDCl3 ……………………………………………………………………. 178

B-7. 13C-NMR of 2,5-dioxopyrrolidin-1-yl 2-phenylquinoline-4-carboxylate

(1h) in CDCl3 …………………………………………………………………..... 179

1 B-8. H-NMR of 6′-N-Hexanoylkanamycin A (2a) in D2O ………………………..…. 180

13 B-9. C-NMR of 6′-N-Hexanoylkanamycin A (2a) in D2O …………………..……… 181

1 B-10. H-NMR of 6′-N-Octanoylkanamycin A (2b) in D2O …………………...... 182

13 B-11. C-NMR of 6′-N-Octanoylkanamycin A (2b) in D2O ………………………… 183

1 B-12. H-NMR of 6′-N-Decanoylkanamycin A (2c) in D2O ……………….…...... 184

13 B-13. C-NMR of 6′-N-Decanoylkanamycin A (2c) in D2O …………….…………... 185

1 B-14. H-NMR of 6′-N-Dodecanoylkanamycin A (2d) in CD3OD ……….……..…… 186

13 B-15. C-NMR of 6′-N-Dodecanoylkanamycin A (2d) in CD3OD ……….…………. 187

1 B-16. H-NMR of 6′-N-Tetradecanoylkanamycin A (2e) in CD3OD ………………… 188

13 B-17. C-NMR of 6′-N-Tetradecanoylkanamycin A (2e) in CD3OD ………………... 189

1 B-18. H-NMR of 6′-N-Hexadecanoylkanamycin A (2f) in CD3OD ………………… 190

13 B-19. C-NMR of 6′-N-Hexadecanoylkanamycin A (2f) in CD3OD ………………... 191

1 B-20. H-NMR of 6′-N-Octadecanoylkanamycin A (2g) in CD3OD ……………….... 192

13 B-21. C-NMR of 6′-N-Octadecanoylkanamycin A (2g) in CD3OD ……………...… 193

B-22. 1H-NMR of 6′-N-(2-phenyl-4-quinolinecarbonyl)kanamycin A

(2h) in D2O …………………………………………………………………….. 194

B-23. 13C-NMR of 6′-N-(2-phenyl-4-quinolinecarbonyl)kanamycin A

(2h) in D2O ………………………………………………………………….... 195

1 B-24. H-NMR of 6′-N-(1-pyrenebutanoyl)kanamycin A (2i) in D2O …………….…. 196

13 B-25. C-NMR of 6′-N-(1-pyrenebutanoyl)kanamycin A (2i) in D2O …………….... 197 xv

1 B-26. H-NMR of 6′-N-(fluorescein)kanamycin A (2j) in D2O …………………...…. 198

13 B-27. C-NMR of 6′-N-(fluorescein)kanamycin A (2j) in D2O ……………………... 199

1 B-28. H-NMR of 6′-N-hexadecanoyltobramycin (3f) in CD3OD ………………….... 200

13 B-29. C-NMR of 6′-N-hexadecanoyltobramycin (3f) in CD3OD ………………..…. 201

B-30. 1H-NMR of 6′-N-Hexanoylkanamycin A (2a) (neutral form)

in D2O …...... 202

B-31. 1H-1H COSY of 6′-N-Hexanoylkanamycin A (2a) (neutral form)

in D2O …………………………………………………………………………... 203

B-32. 1H-1H COSY of 6′-N-Hexanoylkanamycin A (2a) (neutral form)

zoomed in D2O ……………………………………………………………….... 204

C-1. Inhibition of Cx26 dependent growth complementation by

kanamycin derivatives ……………………………………………………..…… 206

C-2. Inhibition of Cx43 dependent growth complementation by

kanamycin derivatives …………………………………………………………………. 207

1 C-3. H-NMR of 6″-O-(4-methoxyphenyl)kanamycin A (11a) in D2O …………...…. 208

13 C-4. C-NMR of 6″-O-(4-methoxyphenyl)kanamycin A (11a) in D2O ……………... 209

1 C-5. H-NMR of 6″-O-(4-methylphenyl)kanamycin A (11b) in D2O ………………... 210

13 C-6. C-NMR of 6″-O-(4-methylphenyl)kanamycin A (11b) in D2O ……………….. 211

1 C-7. H-NMR of 6″-O-phenylkanamycin A (11c) in D2O …………………………… 212

13 C-8. C-NMR of 6″-O-phenylkanamycin A (11c) in D2O …………………………... 213

1 C-9. H-NMR of 6″- O-(4-chlorophenyl)kanamycin A (11d) in D2O ………………... 214

13 C-10. C-NMR of 6″-O-(4-chlorophenyl)kanamycin A (11d) in D2O ………………. 215

1 C-11. H-NMR of 6″-O-(4-nitrophenyl)kanamycin A (11e) in D2O ………………….. 216 xvi

13 C-12. C-NMR of 6″-O-(4-nitrophenyl)kanamycin A (11e) in D2O …………………. 217

1 C-13. H-NMR of 6″-O-(4-(1,1′-biphenyl))kanamycin A (11f) in D2O …………….... 218

13 C-14. C-NMR of 6″-O-(4-(1,1′-biphenyl))kanamycin A (11f) in D2O ……..……… 219

C-15. 1H-NMR of 6″-O-(4-(1H-benzo[d]imidazol-2-yl)phenyl)kanamycin A

(11g) in D2O …………………………………………………………………….. 220

C-16. 13C-NMR of 6″-O-(4-(1H-benzo[d]imidazol-2-yl)phenyl)kanamycin A

(11g) in D2O ……………………………………………………………………. 221

C-17. 1H-NMR of 6 -N-(6-hydroxy-2-naphthoyl)kanamycin A

(12a) in D2O ………………………………………………………………….. 222

C-18. 13C-NMR of 6 -N-(6-hydroxy-2-naphthoyl)kanamycin A

(12a) in D2O ………………………………………………………………….. 223

C-19. 1H-NMR of 6 -N-(6-methoxy-2-naphthoyl)kanamycin A

(12b) in D2O ………………………………………………………………….. 224

C-20. 13C-NMR of 6 -N-(6-methoxy-2-naphthoyl)kanamycin A

(12b) in D2O ………………………………………………………………….. 225

xvii

LIST OF SCHEMES

Scheme

1-2. Synthesis of antifungal AK K20 ……………………………………………….…… 9

1-2. Synthesis of 4″ and/or 6″ modified antifungal AK ………………………………... 11

2-1. Synthesis of amphiphilic kanamycin ……………………………………………………… 39

3-1. Synthesis of amphiphilic aminoglycosides ……………………………………………….... 69

4-1. Synthesis of 6″ aryl modified kanamycin A derivatives …………………….…… 109

4-2. Synthesis 6ʹ aryl modified kanamycin derivatives ……………………………….. 110

xviii

ABBREVIATION

AG: Aminoglycoside

AK: Amphiphilic kanamycin

ATP: Adenosine triphosphate

COSY: Correlation spectroscopy

Cx: Connenexin

DCF-DA: 2’,7’-dichlorofluorescein diacetate

DCF: 2’,7’-dichlorofluorescein

DCM: Dichloromethane

DMEM: Dulbecco’s Modified Eagle Medium

DMF: Dimethylformamide

DMSO: Dimethyl sulfoxide

Equiv: Equivalent

FBS: Fetal bovine serum

FDA: Food and drug administration

FHB: Fusarium head blight

GJC: Gap junction channel

HC: Hemichannel

IC50: Concentration at which 50 % of the cells were dead

LB: Lysogeny broth

PDB: potato dextrose broth

MIC: minimum inhibitory concentration

MOPS: 3-(N-morpholino)propanesulfonic acid

MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide xix

NHS: N-hydroxy succinimide

NMR: Nuclear magnetic resonance

PBS: Phosphate-buffer saline

PI: propidium iodide

ROS: reactive oxygen species

SAR: Structure-activity relationship

TsCl: Toluenesulfonyl chloride

CHAPTER-1

GENERAL INTRODUCTION

1.1 Aminoglycosides

Aminoglycosides (AGs) are extensively used antibiotics in clinical practice for

more than seven decades. AGs are bactericidal and have the inhibitory effect on both

gram-positive (G+) and gram-negative (G-) bacteria.1 Isolated from Streptomyces griseus

in 1944, streptomycin is the first reported AG antibiotic.2 Streptomycin was the first

antibiotic used to treat Mycobacterium tuberculosis after approval by the FDA in 1946. In

addition to Mycobacterium, streptomycin is effective against a wide range of bacterial

strains. Selman Abraham Waksman won the novel prize in 1952 for this discovery.3

Neomycin, kanamycin, gentamicin, tobramycin, sisomicin were subsequently isolated aminoglycosides antibiotics from natural sources, either bacteria or fungi.4-8 AGs are

categorized into two groups based on the substitution position of the 2-deoxystreptamine

ring (Figure 1-1). Kanamycin A, kanamycin B, tobramycin, etc. with substitution at 4 and

6 positions of 2-deoxystreptomine are 4,6-disubstituted AGs. Similarly, Neomycin B,

neomycin C, paromomycin, Ribostamycin, etc. with substitution at 4 and 5 positions of

2-deoxystreptamine are 4,5-disubstituted AGs.9

NH HO HN 2 O H O N NH HO 2 NH OHC 4 2 2 NH HO 1 NH HO 2 O OH 5 6 OH HO H 2- Deoxystreptamine HO O N HO Streptomycin R1

NH2 HO O HO R3 O NH2 2 H2N R NH O NH R1 2 O 2 H HO O N R4 O OH HO O OH O O OH HO 3 OH R NH2 H2N HO HO R1 R2 R3 R4 O R2 OH OH NH OH 3 Kanamycin A 2 R1 R2 R H Kanamycin B NH2 OH NH2 NH H CH2NH2 Tobramycin NH OH OH H Neomycin B 2 2 CH NH H H Neomycin C NH2 2 2 Dibekacin NH2 H H Amikacin OH OH OH AHB Paromomycin OH H CH2NH2 H Arbekacin NH2 H AHB

AHB: (S)-(4)-Amino-2-hydroxybutyryl

Figure 1-1. Structure of streptomycin, 2-deoxystreptamine, and kanamycin and neomycin class aminoglycosides

AGs bind to the aminoacyl-tRNA recognition site of the16S rRNA of the 30S

ribosomal subunit associated with the cell membrane.10-11 This permanent binding of AGs

disrupts the protein synthesis in the bacterial cell membrane, ultimately leading to

bacterial cell death. The emergence of bacterial resistance jeopardized the clinical use of

AGs. Cell membrane modification, efflux pump, modification of the ribosomal site, and

enzymatic modification are resistance mechanisms developed by bacteria against 3

AGs.10, 12-15 Among these, aminoglycoside modifying enzymes (AMEs) ruined the

effectiveness of the antibiotic mostly.12, 16 AMEs modify hydroxyl and/or amine groups present on the AGs, resulting in the poor binding of AGs to the rRNA. Acetyltransferase

(ACC), adenyltransferase (ANT), and phosphotransferase (APH) are the three groups of

AMEs found in the bacteria.16 ACC, the largest group of AMEs, catalyze acetyl

coenzyme-A dependent acetylation of the amino group. APH, the second largest group of

AMEs, catalyze the adenosine triphosphate (ATP) dependent phosphorylation of the

hydroxyl group. ANT, the smallest group among three, catalyze the adenylation of

hydroxyl and amino groups in the presence of ATP. Although AMEs are capable of

modifying both 4,6-disubstituted and 4,5-disubstituted AGs, 4,6-disubstituted AGs are

more affected (figure 1-2).15 While the number is continuously increasing, there are more

than 50 AMEs reported.17

AAC(6'), APH(2") ANT(4') NH ANT(4') 2 AAC(3) O NH2 AAC(3) HO APH(3') HO O AAC(1) NH2 HO H2N HO O NH NH2 HO 2 O HO OH O NH O APH(3') HO 2 AAC(2') O OH O OH HO O AAC(6'), APH(2"), ANT(2") OH H2N NH2

H2N HO HO O Neomycin B Kanamycin A

Figure 1-2. Aminoglycoside modifying enzymes (AMEs) for the modification of

different groups of kanamycin A and neomycin B 4

To revive the antibiotic activity, natural AGs were modified at different positions.

The main aim was to make novel semisynthetic AG which still bind to the rRNA, but will

be a poor substrate to AMEs. Amikacin, netilmicin, isepamicin, dibekacin, and arbekacin

are some examples of semisynthetic AGs.1, 10, 18-19 Amikacin and arbekacin were the most

successful compounds from these series. Unfortunately, some of the bacteria were able to

develop resistance against these new drugs as well.20-21 To combat this issue, structural

modification of AGs, which alter the mode of action, may provide a brighter future for

these drugs.

1.2 Antibacterial Amphiphilic Aminoglycosides

Amphiphilic aminoglycoside is a new class of AGs obtained by the introduction of a hydrophobic, alkyl or aryl, group into hydrophilic AGs. Amphiphilic neomycin is the first synthetic antibacterial amphiphilic aminoglycoside.22 Among the series of 5‴

modified neomycin B derivatives, amphiphilic neomycin integrated with hexadecyl and

octadecyl linear chain had excellent antibacterial activity, including methicillin-resistant

Staphylococcus aureus (MRSA), vancomycin resistance Enterococci (VRE), and AGs

resistance strains. The antibacterial activity of these compounds came from the

permeabilization of the cell membrane instead of binding to tRNA and disrupting protein synthesis like conventional AG.23 In addition to amphiphilic neomycin, amphiphilic aminoglycosides derived from paromomycin, neamine, tobramycin, and kanamycin B

with alkyl or aryl modification were reported for their excellent antibacterial activity

towards resistant and susceptible strains.24-30 Even though these membrane targeting

compounds have excellent activity, there is no drug candidate in the line for clinical

approval. 5

1 NH R 3 - COO O [CF3 ]4 HO O HO HO R3O NH2 NH3 H2N NH3 O NH O NH O H O 2 HO 3 N O OH OR4

R2 R3, R4 = alkyl or aryl group NH3 O OH HO O - NH2 HO [CF3COO ]5 R5 HO NH3 H3N HO O O NH HO 3 R1 O R5 S Neomycin B NH2 R6 HO O Paromomycin OH Tobramycin H OH Kanamycin B OH H N R2 = Alkyl/aryl group 2 R6 = alkyl group

Figure 1-3. Structure of antibacterial amphiphilic aminoglycosides

1.3 Antifungal Amphiphilic Aminoglycosides

Pathogenic fungi have an enormous impact on plant and animal life. Fungal

infections cost billions of dollars in the health sector and food production. 31

Magneporthe oryzae, Botrytis cinerea, Puccinia spp., and Fusarium spp. are the most

detrimental fungal pathogens in the agricultural loss.32 Fusarium head blight (FHB),

primarily caused by the Fusarim graminearum is one of the most devastating diseases in small grain cereal.33 FHB not only reduces the quality of the grain but also affects human health by the production of mycotoxins such as deoxynivalenol (DON).34-35 It is

estimated that there are more than 1.7 billion cases of fungal infections in humans each

year. Fungal infections can range from mild mucocutaneous infections to life-threatening

invasive infections. Aspergillis fumigatus, C. albicans, Candida auris, Cryptococcus

neoformans, and Pneumocystis pneumonia are the most encountered human fungal 6

pathogens.36 Fungal infection can occur in any human, mostly affecting those with immunosuppressant condition like HIV/AIDS, cancer, and organ transplantation.37-38

Annual death of more than 1.6 million people related to fungal infection is similar to

tuberculosis and at least three-fold more than malaria.31, 36 Even though fungal infection

in humans is a serious health problem, it is often neglected compared to viral, bacterial,

and malarial infections.39 In 2017, the disease caused by Candida and Aspergillus alone

cost $4.2 billion, out of a total $7.2 billion loss to the fungal infection in the United

States.40 Candida auris, isolated for the first time in 2009, is an emerging human fungal

pathogen with resistance to commonly practiced antifungal drugs.41-43 Up to now,

Candida auris has been reported in 33 countries. With the mortality rate of more than

68% and recent reports of hospital and nursing home outbreaks, this superbug is a big

threat to global health.

Azoles are the first-line medication for the treatment of fungal infections in humans.44-45 Fluconazole, voriconazole, itraconazole, and posaconazole are commonly

used drugs. Throughout use, fungi start to develop resistance against these drugs, making

them ineffective in treating fungal infections.46-47 One of the reasons behind growing

fungal resistance is the use of azoles both in farming practices and human medical care.

In addition to resistance issues, azoles interact with cytochrome P450S enzymes in

humans, causing adverse effects.45 In this regard, amphiphilic kanamycin (AK) is a new

class of compounds with wide-spectrum antifungal activity.48 The AGs have been in

clinical practice for more than seventy years. Therefore, AKs are one step ahead of others

in the race for novel antifungal drugs. 7

Antifungal AK synthesized by the introduction of an alkyl chain at 4″ position of

kanamycin analogcatalyzes showed antifungal activity against both plant and human

fungal pathogens including azole-resistant strains.49 Three compounds with C-4, C-8, and

C-12 linear alkyl chain at 4″ position were synthesized by the glycorandomization of neamine and glucose derivatives. Among these, compound FG08 with an octyl chain had

the best antifungal activity. A series of compounds with octyl/hexyl chain at different

hydroxyl groups were synthesized and tested for their bioactivity to explore the best

modification site for antifungal activity.50 The results showed that the 4″ position is the

best alkyl modification site, and the octyl chain is still an optimal carbon chain length.

FG06, with the octyl chain at 6″ position, was the second most active compound after

FG08 and FG03. Despite the excellent antifungal activity of these lead compounds,

multiple synthetic steps, and poor (less than 1%) overall yield limit the further study of

these compounds for potential application in agriculture and animals.

[B] [A] ≥ 500 NH FG13 NH3 4' 3 - 4' - [Cl ]4 O HO O HO [Cl ]4 HO 1' HO 1' NH3 NH3 3' 4 3' H N 4 H3N 3 O 62.5 O 1 NH ≥ 500 1 NH3 FG05 HO 3 HO FG12 5 6 5 6 O O 6" 1" 6" ≥ 500 1" OH FG10 2" O HO 2" O HO OH OC8H17 3" 3" 4" HO 4" HO 250 FG09 62.5 62.5 FG03 FG07 MIC (µg/mL) against F. graminearum

Figure 1-4. Structure of FG compounds. [A] FG08, [B] FG compounds with their

minimum inhibitory concentrations. 8

AK K20, synthesized from kanamycin A and octane sulfonyl chloride in three steps, with more than 80% overall yield effectively overcame the poor yield issue of

FG08. 51 The minimum inhibitory concentration of K20 was similar to FG08 against both human and plant fungal pathogens. In addition to solo antifungal activity, K20 also had synergistic antifungal activity towards human pathogens Candida and

Cryptococcus species and plant pathogen Fugarium graminearum when treated in combination with commercially available antifungal azoles.52-53 K20 was also tested in the field for its antifungal activity alone and with half leveled agro-fungicides.53 The result showed the excellent activity of K20 against fusarium head blight on barley when used solely or in combination. Despite the excellent broad-spectrum antifungal activity, synergistic activity in in-vitro and field study, some issues are associated with K20. The first of these is the cost of production, which is ten times more than the marketed agrofungicides. The second is the stability of the octane sulfonyl group, which is labile at acidic or basic pH of the solution. 9

Scheme 1-1. Synthesis of antifungal AK K20.

NH2 NHBoc O O HO Boc O HO HO 2 HO NH2 NHBoc HO MeOH, H2O HO O NH2 O NHBoc HO HO O O 6"OH 6"OH HO O O OH HO OH H2N BocHN

Kanamycin A B4K

NH3 O HO - HO NH3 [Cl ]4 1. C8H17SO2-Cl, py HO O NH HO 2 2. TFA, Ion exchange O 6"O O S HO O OHO C8H17 H3N K20

The antifungal AKs with oxygen as a connector atom between a hydrophobic

group and kanamycin moiety solved the pH instability problem associated with K20.54

These three classes of compounds with 4″ and/or 6″ alkyl modification were synthesized from nucleophilic substitution of alkyl bromide with hydroxyl group followed by column purification. Final compounds were obtained by the reduction of azide, CG50 purification, and then ion exchange (Scheme 1-2) followed by reduction of azide.

Compounds substituted at 4″ or 4″ and 6″ position with C-6, C-7, C-8, and C-9 had shown antifungal activity with little or no antibacterial activity. Among these active compounds, C-6 and C-8 disubstituted compounds displayed superior activity. The transformation of kanamycin A to Azido kanamycin, an intermediate of the reaction, requires sodium azide along with other reagents. Sodium azide is an explosive reagent, especially when used on a larger scale. The explosive nature of sodium azide limits the 10

large-scale synthesis of these compounds. The poor separation of 4″ and 6″ substituted compounds due to similar polarity, and mandatory column purification further delimited the translational value of these compounds. Following this, 4″-6″ disubstituted kanamycins with aromatic groups with antifungal compounds were also reported.55

Regardless of the good antifungal activity of both alkyl or benzyl substituted analogs towards plant and human fungal pathogens, the necessity of sodium azide restricts the synthesis of these compounds on a large scale.

Scheme 1-2. Synthesis of 4″ and/or 6″ modified antifungal AK

N3 NH2 4' O O HO HO HO R-Br, NaH 1' N3 HO NH TfN3, CuSO4(cat.) 3' 4 2 HO DMF HO O N O 1 NH 3 2 H2O:MeOH:DCM(1:3:1) HO HO 6 O 5 O OH 1" 6" OH O O HO OH HO 2" OH 3" 4" N3 H2N

Kanamycin A NH3 - N3 O HO [Cl 4] HO O PMe NH HO 3 OH 3 HO THF, H O N3 2 O NH OH HO 3 O N3 CG50 purification, O R = alkyl group or H HO R' = alkyl group or H O Ion exchange OR O OR HO OR' HO O OR' H3N N3

N3 NH3 O HO 1. R-Br, NaH O - HO HO [Cl 4] N3 DMF HO OH NH3 O N OH 3 O NH HO 2. PMe3 3 O HO THF, H2O O OH 3. O CG50 purification, OR" HO OH Ion exchange O HO OR" N3 H3N

R" =

OMe

Amphiphilic tobramycin, previously reported for its antibacterial activity, also showed antifungal activity towards human and plant fungal pathogens.27, 56 These 12

compounds had optimal antifungal activity with a longer carbon chain (C12 and C14), as

oppose to the optimal carbon chain length of the kanamycin A derived antifungal

compound. Amphiphilic tobramycins were synthesized by the introduction of even

number alkyl chain from C-4 to C-14 group at 6″ position. Notable differences of these compounds from the kanamycin A derived amphiphilic compounds were the connector

atom between the alkyl chain and AG core and the number of amine groups in the AG

core. The compounds derived from kanamycin A had four amine groups with oxygen as a

connector atom, whereas the tobramycin derived compounds had five amine groups with

sulfur as a connector atom. Like amphiphilic tobramycin, kanamycin B derived AK also

had excellent antifungal activity in addition to antibacterial activity. In the case of

kanamycin B derived compounds as well, longer carbon chain with >C14 were the most

active.29 Lead compounds derived from both tobramycin and kanamycin B had better

antifungal activity than FG08 and K20. Tobramycin and kanamycin derivatives with pH

stable sulfur linker atom and better antifungal activity seem to have an edge compared to

K20, but the cost of the AGs itself put them down in terms of cost of production. The

cost of tobramycin and kanamycin B is at least 10-fold more than the kanamycin A used in the production of the K20.

The limitations associated with previously reported antifungal amphiphilic aminoglycosides may be resolved by introducing new analogs with a stable connector group between the hydrophilic and hydrophobic groups and made up of cheaper starting material.

1.4 Aminoglycosides as a Connexin Hemichannel Inhibitor

Hemichannels (HCs) are homomeric or heteromeric hexamer of connexin

isoforms.57-58 There are twenty-one connexin genes reported in human genomes, which

express different connexins isoforms.59 Connexins are named as Cx with a numerical

suffix, corresponding to the molecular weight in kilodalton.60 For example, connexin with

43 KDa molecular weight is named as Cx43. The connexins can form a variety of HCs

and gap junction channels (GJCs) with different regulations and permeability.61-66

Connexins have four hydrophobic transmembrane domain (M1-M4), a cytoplasmic loop

(CL) between TM2 and TM3, two extracellular loops (E1 and E2), a cytoplasmic C- terminus (CT) and N-terminus (NT) (Figure 1-6).61-62, 64, 66-69 The C-terminal of connexin can be phosphorylated and mediate interaction with other proteins whereas the N- terminal play roles in channel functioning and gating.60, 70-73 External loop of connexin

involved during the formation of gap junction channels (GJCs).74

Figure 1-5. Schematic representation of Connexin monomer (top), hemichannel (HC), and gap junction channel (GJC)

GJCs formed by the head to head docking of two connexin HCs play a crucial role in the cellular growth, embryogenesis, homeostasis, and electrical transduction by allowing the intercellular exchange of ions, metabolites, and small molecules.61, 75 It was believed that HC in normal conditions either remain closed or docked with each other HC to form gap junction channels. Recently, it was found that these hemichannels can open 15

involuntarily and let the exchange of ions and cellular metabolites such as ATP,

glutamate, NAD+ in to the intercellular milieu in different physiological conditions.76

Uncontrolled opening or activation of HCs contributes to cell damage in many genetic

and acquired diseases, including ischemic damage of the heart (infarct), brain (stroke)

and kidneys, some forms of deafness, and neurodegenerative diseases.60, 77-82 Inhibitors

selective to HC can be crucial in the treatment of deafness, neurodegenerative diseases,

heart problems, stroke, and cancer.83-94

Many of the known connexin inhibitors are n-alkanols, volatile anesthetics, and

blockers of other channels, altering many other cellular processes95-97. The rationally

designed connexin-targeted peptide inhibitors are more promising; however, they primarily

target GJCs and their potential translational value as useful clinical agents are still

unclear.98-103 On the other hand, gentamicin and streptomycin had excellent inhibitory

activity towards connexin HCs.104-106 Interestingly, gentamicin had inhibitory activity

towards Cx26, Cx43, and Cx46 HCs but did not affect GJCs, making this selective towards

HCs. 106 More commercial aminoglycosides were tested for their activity to Cx26, Cx43,

and Cx46 HCs.107 These aminoglycosides were more effective in inhibition of Cx26

compared to the other tested connexin HCs. We previously reported 4″-6″ disubstituted

compounds with excellent Cx26 HCs inhibitory activity and no toxicity towards human

cells.108-109 The lead from these compounds was about four-fold more active than

kanamycin. AGs are in clinical practice for more than seven decades with well-known

pharmacokinetics. In this scenario, AGs may be the right pharmacophore to modify in

order to find the connexin HCs selective inhibitors. 16

1.5 Specific Aims of the Work

The common objective of the work was to explore the translational values of

kanamycin derivatives with antifungal and connexin inhibitory activities. To achieve this broad goal, previously reported leads were optimized in terms of their stability and

bioactivity.

The first aim of the work was a cost-effective and scalable synthesis of

antifungal AKs with a stable linker group between the hydrophobic group and the

kanamycin core. For this, a new library of 6″ alkyl modified AKs with nitrogen or sulfur

as a connector atom at the 6″ position was synthesized, and the leads were identified from

SAR study. The cost of the newly synthesized 6″ modified compound was not

competitive with marketed antifungals; therefore, a series of AKs with modification at an

alternative site were explored. Fatty acids are cheaper than the alkyl amine and alkyl

thiol, and the 6ʹ modification can be achieved in one step from kanamycin A. Thus, the

cost of 6ʹ modified AKs was lower than the 6″ modified AKs and comparable to

marketed antifungal compounds.

The second part of the work was aimed to explore the factors controlling fungal selectivity of the AKs. Kanamycin A derived antifungal amphiphilic compounds were

selective towards fungal strains relative to bacteria and human cells. Fluorescent analogs

were synthesized by the introduction of fluorophore at the 6″ or 6ʹ position of kanamycin.

Fluorescence imaging was used to monitor the cellular uptake of the fluorescent analogs

into different types of cells.

The last aim of the work was to explore a kanamycin derivative which can

selectively inhibit the Cx43 HC. Commercial AGs had excellent inhibitory activity 17

towards Cx26 and Cx43 HCs, though with selectivity for Cx26 HC compared to Cx43

HC. Also, 4″ and 6″ modified kanamycin A compounds had improved activity towards

Cx26 HC compared to parent compounds, but they were not tested for Cx43 HC inhibitory activity. A library of kanamycin derivative with modification at the 6ʹ or 6″ position was synthesized and tested for their inhibitory activity towards Cx26 and Cx43

HCs. In addition to newly synthesized compounds, previously reported 4″ and 6″ disubstituted kanamycin are also tested for their potential selectivity towards Cx43 HC.

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CHAPTER-2

SCALABLE AND COST-EFFECTIVE TOSYLATION-MEDIATED SYNTHESIS OF

ANTIFUNGAL AND FUNGAL DIAGNOSTIC 6″-MODIFIED

AMPHIPHILIC KANAMYCINSa

2.1 Abstract

Amphiphilic kanamycins bearing hydrophobic modifications at the 6″ position have attracted interest due to remarkable antibacterial-to-antifungal switches in bioactivity. In this report, we investigate a hurdle that hinders practical applications of these amphiphilic kanamycins: a cost-effective synthesis that allows the incorporation of various connecting functionalities to which the hydrophobic moieties are connected to the kanamycin core. A cost-effective tosylation enables various modifications at the 6″ position, which is scalable to a ninety-gram scale. The connecting functionalities, such as amine and thiol, were not the dominant factor for biological activity. Instead, the linear chain length played the decisive role. Amphiphilic kanamycin attached with tetradecyl (C14) or hexadecyl (C16) showed strong antifungal and modest antibacterial activities than with shorter chains (C6-C10). However, increases in chain length were closely correlated with an increase in HeLa cell toxicity.

aAdapted with permission from (Subedi, Y.P.; Pandey, U.; Alfindee, M.N.; Montgomery, H.; Roberts, P.; Wight, J.; Nichols, G.; Grilley, M.; Takemoto, J.Y. and Chang, C.-W.T. “Scalable and cost-effective tosylation-mediated synthesis of antifungal and fungal diagnostic 6″-Modified amphiphilic kanamycins.” Eur. J. Med. Chem., 2019, 182, 111639). Copyright © 2019, Elsevier Masson. 35

Thus, a compromise between the antimicrobial activities and cytotoxicities, for optimal efficacy of amphiphilic kanamycins may contain chain lengths between C8 and C12.

Finally, the described synthetic protocol also allows the preparation of a fluorescent amphiphilic kanamycin selective toward fungi. 36

2.2 Introduction

Amphiphilic kanamycin (AK) such as those synthesized from kanamycin A, kanamycin B and tobramycin–all with hydrophobic moieties attached at the 6″-position– has attracted great interest due to their broad spectrum antibacterial and antifungal activities 1-5. The reported synthetic approaches have allowed studies on structure- activity relationships (SARs), including the effects of connecting functionalities, such as thiol, sulfone, amide, ether, and 1,2,3-triazole) and the optimal chain length of the linear hydrophobic groups. Despite the advances, cost-effective and scalable production of the lead compounds has not been satisfactorily resolved. In addition, there is no AK bearing an amine as the connecting functionality. Amines can amend the solubility or bioavailability of the AK (especially for those with longer (>C14) linear hydrophobic chains) by bringing additional hydrogen bonds or cations. To address these shortcomings, we decided to employ kanamycin sulfate as the starting material and explore toluenesulfonyl as the connecting functionality in the chemical synthesis of AK- carrying amino hydrophobic moieties attached at the 6″-position. Commercially available kanamycin sulfate (containing >95% kanamycin A) is more cost-effective compared to kanamycin B, tobramycin, or other kanamycin-class aminoglycosides

(Figure 2-1).

NH2 O HO 2 R H N R1 2 O NH HO 2 O OH O HO OH H2N R1 R2 Kanamycin A OH OH Kanamycin B NH2 OH Tobramycin NH2 H

Figure 2-1. Structure of kanamycin class aminoglycosides

2.3 Rationale, Design and Synthesis

To implement modifications at the hydroxyl groups of kanamycin, the amino groups need to be protected or masked as azido groups. The former is commonly achieved using carbamate-type protecting groups, e.g. t-butoxycarbonyl (Boc) or carboxybenzyl (Cbz) groups. The latter can be done via amine/azide transformation. We favor the use of Boc groups for two reasons. First, the reagent (Boc2O) is inexpensive and much safer to handle as compared to azide needed for the amine/azide transformation. Second, Boc groups can be removed without trace using TFA, whereas removal of Cbz requires hydrogenolysis using a costly and hazardous metal catalyst. We have previously reported the large scale synthesis of Boc-protected kanamycin (300g scale) without the need of column purification6. The next hurdle is the regioselective incorporation of a hydrophobic group at the 6″-position. This is conducted by selectively converting the 6″-OH into a leaving group, followed by nucleophilic substitution. A bulky 2,4,6-triisopropylbenzenesulfonyl (TIPBS) chloride is commonly used to enable selective incorporation at the 6″-OH7-8. However, TIPBSCl is not cost-effective and usually needs to be employed in excess amount (5 equiv.). We believe that 38

toluenesulfonyl (Ts) chloride is a better option. After several attempts, we achieved comparable yields as those obtained using TIPBSCl but with only 2.5 equiv. of TsCl to generate compound 2 (Scheme 1). Furthermore, we demonstrated that the reaction can be conducted at larger scales (ca. 90 g scale). In contrast, the reported synthesis using

TIPBSCl was often conducted on a scale less than 5 g. The tosylation site was confirmed from the 1H-1H COSY of compound 3.

Following the successful synthesis of tosylated kanamycin derivative, nucleophilic substitution using linear alkylamines (hexyl, octyl, decyl, dodecyl, tetradecyl and hexadecyl amines) followed by column fractionation and deprotection of Boc groups provided the desired products. To compare similar compounds as reported by others, we also used linear alkyl thiols (hexyl, octyl, decyl, dodecyl, tetradecyl, and hexadecyl thiols) as the nucleophiles. Without purification using column chromatography, the Boc groups of the substitution products were removed using TFA and furnished the final AK ready for biological assay. In addition, we synthesized a fluorescent member, 7 using an amide as the connecting functionality. Compound 7 enables fluorescence-based evaluation of microbe selectivity (fungi vs. bacteria), which will be discussed later.

Finally, it is important to mention that the tosylated kanamycin derivative can also lead to the synthesis of AK with ether, amide, or sulfone connecting functionalities using established protocols3, 9.

Scheme 2-1. Synthesis of amphiphilic kanamycin

NHBoc NHBoc NH3 O HO O HO HO O - HO HO NHBoc NHBoc HO H N [Cl ]4 OH Ts-Cl OH 1. TFA, DCM OH 3 O NHBoc O NHBoc O NH HO py HO 2. Ion exchange HO 3 O O O OH OTs OTs O HO O O HO OH OH HO OH BocHN BocHN H3N 1 2 R-SH, CsCO3 3 DMF, R. T. 54 % 95 % R-NH 2 o Dioxane, 100 C 1. TFA, DCM 1. TFA, DCM 2. CG50 purification, 2. CG50 purification, Ion exchange (Cl-) Ion exchange (Cl-) NH NH3 3 - - O HO O HO [Cl ]4 HO [Cl ]5 HO NH NH3 OH 3 OH O O NH NH3 HO 3 HO O O SR NH2R O HO O HO OH OH H N H3N 3

R R'

4a C6H13 56% 5a C6H13 71% 4b C H17 45% 8 5b C8H17 54% 4c C10H21 65% H 5c C10 21 45% 4d C12H25 59% C H 5d 12 25 61% 4e C H 47% C H 14 29 5e 14 29 43% 4f C16H33 20% 5f C16H33 31%

O NHBoc O NH3 O 1. N O - HO HO [Cl ]4 HO R'" HO NHBoc O NH3 1. NaN3, DMF OH OH o O NHBoc 2. TFA, DCM O NH 80 C, 24 hrs HO HO 3 O 2 O O NH 3. CG50 purification, 2. H2, Pd/C 2 NH R"' HO O ion exchange 7 HO O MeOH/HOAc 6 OH OH BocHN H3N 48 % R'" = O O OH

23 % 40

2.4 Results and Discussion

2.4.1 Antifungal activity of amphiphilic kanamycin

The antifungal activity of newly synthesized AK was tested against a panel of fungi, including Candida albicans 64124 (human pathogen, azole-resistant), C. albicans

MYA2876 (human pathogen, azole susceptible), C. albicans B-311 (human pathogen),

C. rugosa 95-967 (human pathogen), C. parapsolis Cas08-0490 (human pathogen, azole-resistant), C. tropicalis 95-41 (human pathogen), Cryptococcus neoformans H99

(human pathogen), C. neoformans VR-54 (human pathogen), Rhodotorula pilimanae

(ATCC 26423, plant pathogen), and Fusarium graminearium B4-5A (plant pathogen), using K20 as the control. The minimum inhibitory concentrations (MICs) were determined (Table 1). From the MICs, AKs bearing tetradecyl (C14) and hexadecyl

(C16) were most potent regardless of the connecting functionalities (amine and thiol).

This is consistent with those thiol analogs reported by others. Interestingly, K20 compounds carrying octanesulfonyl groups are only slightly less active than the AK bearing tetradecyl (C14) and hexadecyl (C16) groups, while those with octyl or decyl chains are much less active, especially against Candida sp. In addition, strains of the genuses Cryptococcus, Rhodotorula, and Fusarium become more prone to the increase in chain length, as compounds 4b,c and 5b,c attached with octyl or decyl groups started to show increased antifungal activity. Based on these findings, it is concluded that the connecting functionality does not play a key role in level of antifungal activity. Rather, the chain length and the strain of fungi are the main factors that govern the antifungal activity of AK with 6″-modification. 41

Table 2-1. Antifungal activity of AK[a]

Compound A[b] B[b] C[b] D[b] E[b] F[b] G[b] H[b] I[b] J[b]

3 >256 >256 >256 >256 >256 >256 >256 >256 128 128

4a >256 >256 >256 >256 >256 >256 128 128 128 64

4b 256 >256 >256 >256 >256 >256 16 16 16 32

4c 128 >256 256 256 256 >256 8 8 8 32

4d 16 64 32 16 32 32 2 2 2 2

4e 4 8 8 4 4 4 2 2 2 2

4f 4 8 4 4 4 4 2 2 2 2

5a >256 >256 >256 >256 >256 >256 128 64 32 256

5b 128 256 >256 256 >256 >256 128 64 32 128

5c 64 64 128 64 128 128 8 8 4 32

5d 16 16 32 16 32 32 2 2 2 4

5e 4 4 8 8 16 4 2 2 2 2

5f 4 4 4 4 4 4 2 2 2 2

7 128 256 ND[c] ND ND ND 64 64 32 32

K20 16 16 16 32 16 16 4 4 4 8

Kan A ≥256 ≥256 ≥256 ND ND ND ND ≥256 ≥256 ≥256

[a] Unit: µg/mL; [b] A: C. albicans 64124, B: C. albicans MYA2876, C: C. albicans

B-311, D: C. rugosa 95-967, E: C. parapsolis Cas08-0490 (azole resistant), F: C. tropicalis 95-41, G: C. neoformans H99, H: C. neoformans VR-54, I: R. pilimanae

(ATCC 26423), J: F. graminearium B4-5A, [c] ND: Not determined.

2.4.2 Antibacterial activity of amphiphilic kanamycin

The antibacterial activity of AK was also tested against a panel of bacteria, including Escherichia coli (ATCC 25922, Gram negative, G-), Staphylococcus aureus

(ATCC 25923, Gram positive, G+), S. aureus (ATCC 33591, MRSA), and S. aureus

(ATCC 43300, MRSA) using kanamycin A and vancomycin as the controls. The results showed that these AK exerted only modest antibacterial activity even for those with C14 or C16 chains, and again the connecting functionalities did not have obvious effect.

Nevertheless, the antibacterial SAR is similar to the antifungal SAR implying the lack of selectivity between antibacterial and antifungal activity. Interestingly, compound 3 with its toluenesulfonyl group displayed an activity profile like kanamycin A; it was active against aminoglycoside susceptible strains while being inactive against MRSA.

Table 2-2. MICs of AK against bacterial strains[a]

Compound A[b] B[b] C[b] D[b] Compound A[b] B[b] C[b] D[b]

3 16 32 >256 >256 5a 128 256 >256 >256

4a 256 64 >256 >256 5b 128 128 256 256

4b 256 128 >256 128 5c 64 64 64 32

4c 64 128 256 64 5d 16 32 32 16

4d 16 64 64 32 5e 16 16 16 16

4e 32 16 16 16 5f 32 16 32 16

4f 32 16 32 32 7 32 64 >256 >256

Kan A 4 4 >256 >256 Vancomycin ND[c] ND 4 2

[a] Unit: µg/mL; [b] A: E. coli (ATCC 25922), B: S. aureus (ATCC 25923), C: S. aureus

(ATCC 33591) MRSA, D: S. aureus (ATCC 43300) MRSA; [c] ND: Not determined.

2.4.3 Cytotoxicity of amphiphilic kanamycin against HeLa cells

Selected AKs with good antifungal activity were tested for their cytotoxicity towards HeLa cells (Figure 2). The IC50 values of the compounds against HeLa cells are presented in Table 3. AK equipped with 12-carbon chain or less did not show toxicity up to tested concentrations while the AK with a carbon chain of fourteen or longer have shown significant toxicity at 100 µg/mL. These results suggest that it may be necessary to make compromises with less than optimal antifungal activities and cytotoxicities for selecting efficacious AK analogs (such as in the case of K20), for clinical and agricultural applications. 44

[a] Table 2-3. IC50 value of the AK against HeLa cells

Compound IC50 value

4d >100

4e 60.79 ± 3.13

41.56 ± 10.23 4f

5d >100

5e 52.88 ± 5.78

5f 37.73 ± 7.33

7 >100

aUnit = µg/mL

4d 4e 4f 5d 5e 5f 7

100

% Cell viability Cell % 25

0 0.1µg/mL 1.0 µg/mL 10 µg/mL 100 µg/mL

Figure 2-2. Cytotoxicity of AK against HeLa cells.

Treatments for fungal diseases can be challenging due to the emergence of drug- resistant fungi and the unclear distinction of fungal infections from bacterial infections.

The latter can be difficult for physicians due to similar symptoms from bacteria and fungi. Nevertheless, in order to offer prompt therapeutic options, it is pivotal to identify the nature of the microbes that cause the infection since antifungal agents are, in general, inactive against bacteria and vice versa. Current practices for the detection of fungi, such as culture method and PCR may take days to yield the result 10. Amphiphilic kanamycin has been shown to exert a fast membrane permeabilization (within a few minutes) and endocytosis selective toward fungi over bacteria 11. Therefore, we decided to synthesize the fluorescent AK, 7 as described in Scheme 1 for the potential application of offering prompt differentiation of fungi from bacteria. Two strains of fungi, C. albicans 64124

(azole-resistant) and C. albicans MYA2876 (azole susceptible), and two strains of 46

bacteria, E. coli (G-) and S. aureus (G+) were selected for the imaging study. Fungi were treated with compound 7 at 32 µg/mL, which is about ¼ of the MIC. Bacteria were also treated with compound 7 at 32 µg/mL, which is the MIC for E. coli and ½ of the

MIC for S. aureus. The images were taken after 5 and 60 minutes of incubation with compound 7 (Figure 2-3).

From the images, it is clear that compound 7 can quickly stain the fungal yeast

Candida strains even with 5 minutes of incubation and at less than MIC concentrations.

In contrast, no fluorescent staining can be observed for both bacteria (E. coli and S. aureus) after 5 minutes. Only after incubating for 60 minutes, did E. coli and S. aureus begin to show slight fluorescent staining. Since the concentration of 7 employed was not fungicidal, but closer to bactericidal, the relatively strong fluorescence emitted from fungal cells when treated with compound 7 indicates that fluorescent AK analogs like compound 7 can be employed for differential diagnostic detection of fungi vs. bacteria at single-cell imaging resolution. 47

Fungal yeasts treated with 7 Bacteria treated with 7

Figure 2-3. Images of fungi and bacteria incubated with 7. (A, B) C. albicans 64124 treated for 5 min; (C, D) C. albicans 64124 treated for 60 min; (E, F) C. albicans

MYA2876 treated for 5 min; (G, H) C. albicans MYA2876 treated for 60 min; (H, I)

E. coli treated for 5 min; (J, K) E. coli treated for 60 min; (L, M) S. aureus treated for

5 min; (N, O) S. aureus treated for 60 min.

2.5 Conclusion

We have developed a practical and scalable route for the synthesis of 6″-modified

AK that is suitable for the introduction of various connecting functionalities between the kanamycin core and hydrophobic moieties. Two libraries of AK bearing amine and thiol connecting functionalities were synthesized. Similar SARs show that tetradecyl (C14) and hexadecyl (C16) are the optimal chain length to exert broad-spectrum antifungal activity and modest antibacterial activity. Little or no difference in bioactivity displayed by the two libraries containing amine or thiol connecting functionalities suggests a minor role of these functionalities. However, the cytotoxicity evaluation revealed that as chain length increases (≥C14), cytotoxicity also increases. Combining these results, we conclude that AK equipped with longer chain length exert superior bioactivity against fungi and bacteria, but are associated with increased cytotoxicity, making these AKs behave more like non-selective antiseptic agents. In contrast, K20 developed previously in our group represents one of the few examples of AK that best compromises between antifungal/antibacterial activity and toxicity to mammalian cells; it is selectively active toward fungi but inactive against bacteria or mammalian cells. The synthesis of fluorescent compound 7 and its fungal selective properties may pave the way for the development of diagnostic tools that can promptly differentiate fungal infection from diseases of bacterial origin.

2.6 Experimental Section

Materials and Methods. Chemicals purchased from the commercial source were used without further purification unless otherwise mentioned. Pyridine was dried by keeping 49

over calcium hydride for 72 hours and recycled for further use. DMF was dried by treating with a molecular sieve for 72 hours before using for the reaction. 1H-NMR, 1H-

1H COSY, and 13C NMR spectra of the compounds were obtained using a Brucker

AvanceIII HD Ascend-500 at 500 MHz and 125 MHz respectively. Multiplicity of the peaks is reported as s = singlet, d = doublet, t = triplet, m = multiplet, b = broad.

Coupling constants are presented in the hertz (Hz). Emission and excitation spectra of 7 were recorded in SHIMADZu RF-5301PC Spectrofluorophotometer. Emission wavelength was fixed at 520 nm while scanning excitation wavelength and the compounds were excited at 480 nm for emission spectra of 7.

General Procedure for the Synthesis of Compound 4a - 4f. To a solution of 2 (0.104 g, 1 equiv, 0.1 mmol) dissolved in anhydrous dioxane, 3 equiv of alky amine was added and the reaction mixture was refluxed for 24 hrs. Solvent was removed under reduced pressure and the solid residue was washed with 3x10 mL of water. During the washing,

10 mL of water was added to the solid, stirred for 15 minutes, and then filtered. This process was repeated two more times before moving to the next step. After washing, the reaction mixture was loaded onto a silica gel column and eluted with solvents ranging from 100% DCM to 10% MeOH in DCM. The eluted fractions were analyzed by TLC

(5% methanol in DCM), and those that contained desired products were combined and concentrated to provide a yellowish white precipitate. The collected precipitate was re- dissolved in anhydrous DCM (10 mL) and trifluoroacetic acid (TFA) (1 mL) was added.

After being stirred for 6 hrs, solvents were removed, and the residual reaction mixture was loaded onto a column packed with Amberlite CG50 ion exchange resin, and eluted 50

with a gradient of solvent (100% water to 20% NH4OH in water). Pure compound obtained after CG50 purification was further subjected to the ion exchange using anion exchange resin IR410 (Cl- form) to yield the desired amphiphilic kanamycin as a chloride salt.

General Procedure for the Synthesis of 5a - 5f. To a solution of 2 (0.104 gm, 1 equiv,

0.1 mmol) dissolved in dimethylformade (DMF), 5 equiv of alkylthiol and 5 equiv of cesium carbonate was added and the reaction mixture was stirred for 48 hrs at room temperature. Solvent was removed, and the precipitate was washed with water 4x20 mL of water using a Buchner funnel. During the washing, 20 mL of water was added to the precipitate, stirred for 15 minutes, and then filtered. This process was repeated three more times. The precipitate was further washed with 3x10 mL of hexane. The residual solid was dissolved in 10 mL of anhydrous DCM and 1 mL of TFA was added, and stirred at room temperature for 6 hours. Solvents were removed, and the residual reaction mixture was loaded onto a column packed with Amberlite CG50 ion exchange resin, and eluted with a gradient of solvent (100% water to 20% NH4OH in water). Pure compound obtained after CG50 purification was further subjected to the ion exchange using anion exchange resin IR410 (Cl- form) to yield the desired amphiphilic kanamycin as a chloride salt.

6''-O-Toluenesulfonyl-1,3,6',3''-tetra-N-(tert-butoxycarbonyl)kanamycin A (2). A solution of 1,3,6',3''-tetra-N-(tert-butoxycarbonyl)kanamycin A (86.9 gm, 1 equiv, 100 mmol) was dissolved in 1200 mL of anhydrous pyridine by magnetic stirring at room 51

temperature. The reaction flask was then transferred to the ice bath and stirred for 15 minutes. A solution of toluenesulfonyl chloride (TsCl) (47.67 gm, 2.5 equiv, 250 mmol) dissolved in 300 mL of anhydrous pyridine was slowly added to the stirring solution of

1,3,6',3''-tetra-N-(tert-butoxycarbonyl)kanamycin A at 0 oC over the period of an hour.

After completion of the addition of TsCl, the reaction mixture was stirred for additional

30 minutes. The ice bath was removed, and the reaction mixture was stirred for 3 days allowing the temperature to rise slowly to room temperature. The reaction was quenched by addition of methanol (6 mL, 1.5 equiv, 0.15 mol). After being stirred for 30 minutes, the solvents were removed using a rotary evaporator under reduced pressure while keeping the temperature of the water bath below 30 oC. The viscous residue obtained after the removal of the solvent solidified into powder and was washed with 5x500mL water using a Buchner funnel. The resulting pale yellowish powder was loaded in to the silica gel column and purified by eluting with a gradient of solvent (100% DCM to 5%

MeOH in DCM). The compound 2 was obtained as a white solid (56.4 g, 6.4 mmol,

1 54%). H NMR (500 MHz, CD3OD) δ 7.79 (d, J = 8.0 Hz, 2H), 7.45 (d, J = 8.0 Hz, 2H),

5.08 (b, 1H), 5.00 (b, 1H), 4.1 - 4.4 (m, 3H), 3.3 - 3.8 (m, 12H), 3.03 (t, J = 9.5 Hz, 1H),

13 2.48 (s, 3H), 2.0 – 2.1 (m, 1H), 1.4 - 1.5 (m, 37H); C NMR (125 MHz, CD3OD) δ

157.97, 157.82, 156.54, 156.28, 132.99, 129.63 (2C), 127.72 (2C), 101.41, 98.38, 84.34,

79.91, 79.22, 79.01 (2C), 78.78, 75.33, 73.10, 72.52, 70.95, 70.45, 70.33, 70.21, 69.05,

67.71, 55.93, 50.60, 49.37, 40.49, 34.63, 27.53 (3C), 27.49 (3C), 27.45 (6C), 20.31.

+ + ESI/APCI calcd for C45H74N4NaO21S [MNa] : 1061.4464; measured m/e: 1061.4474.

6''-O-Toluenesulfonylkanamycin A (3). To a solution of 2 (0.104 gm, 0.1 mmol) dissolved in anhydrous DCM (5 mL), trifluoroacetic acid was added (0.5 mL). After being stirred at room temperature for 6 hours, the solvent was removed. The crude product was subjected to ion exchange using anion exchange resin IR410 (Cl- form) to yield the desired amphiphilic kanamycin as a chloride salt. The compound was

1 synthesized as white solid (74.5 mg, 0.095 mmol, 95%). H NMR (500 MHz, D2O) δ

7.72 (d, J = 8.5 Hz, 2H), 7.37 (d, J = 8.5 Hz, 2H), 5.49 (d, J = 4.0 Hz, 1H), 4.94 (d, J =

3.5 Hz, 1H), 4.2 - 4.3 (m, 2H), 4.0 – 4.1 (m, 1H), 3.9 – 4.0 (m, 1H), 3.2 – 3.9 (m, 12H),

3.1 - 3.2 (m, 1H), 2.4 - 2.5 (m, 1H), 2.34 (s, 3H), 1.8 - 1.9 (m, 1H); 13C NMR (125 MHz,

D2O) δ 146.75, 130.37, 130.24 (2C), 127.93 (2C), 100.55, 96.25, 83.82, 78.20, 72.53,

72.08, 70.82, 70.81, 70.33, 69.06, 68.69, 67.90, 65.19, 54.72, 49.93, 47.89, 40.43, 27.58,

+ + 20.97. ESI/APCI calcd for C25H43N4O13S [MH] : 639.2547; measured m/e: 639.2543.

6″-(1-Hexylamino)-6″-deoxykanamycin A (4a). The compound was synthesized according to the general procedure and obtained as a white solid (41.9 mg, 0.056 mmol,

1 56%). H NMR (500 MHz, D2O) δ 5.75 (d, J = 4.0 Hz, 1H), 5.08 (d, J = 3.5 Hz, 1H),

4.1-4.2 (m, 1H), 3.3 – 4.0 (m, 15H), 3.1 - 3.2 (m, 1H), 3.03 (t, J = 9.0 Hz, 2H), 2.4 - 2.5

(m, 1H), 1.8 - 1.9 (m, 1H), 1.6 - 1.7 (m, 2H), 1.2 - 1.4 (m, 6H), 0.79 (t, J = 7.0 Hz, 3H);

13 C NMR (125 MHz, D2O) δ 100.4, 98.5, 83.7, 79.7, 74.1, 71.8, 70.9, 70.6, 68.7, 68.2,

68.0, 67.2, 54.6, 50.1, 48.4, 48.3, 47.7, 40.2, 30.4, 28.3, 25.3 (2C), 21.7, 13.2. ESI/APCI

+ + calcd for C24H50N5O10 [MH] : 568.3558; Measured m/e: 568.3576.

6”-(1-Octylamino)-6”-deoxykanamycin A (4b). The compound was synthesized according to the general procedure and obtained as a white solid (35.0 mg, 0.045 mmol, 53

1 45%). H NMR (500 MHz, D2O) δ 5.44 (d, J = 4.0 Hz, 1H), 5.03 (d, J = 3.5 Hz, 1H), 4.1

- 4.2 (m, 1H), 3.1 – 4.0 (m, 16H), 3.02 (t, J = 8.0 Hz, 2H), 2.2 - 2.3 (m, 1H), 1.5 - 1.7 (m,

13 3H), 1.1 - 1.3 (m, 10H), 0.78 (t, J = 6.5 Hz, 3H); C NMR (125 MHz, D2O) δ 100.37,

98.44, 83.72, 79.69, 74.18, 71.84, 70.00, 70.75, 68.71, 68.25, 68.04, 67.21, 54.64, 50.13,

48.42, 48.36, 47.71, 40.33, 31.01, 28.35, 28.20, 28.18, 25.66, 25.37, 21.79, 13.40.

ESI/APCI calcd for C26H54N5O10: 596.3871; measured m/e: 596.3864.

6″-(1-Decylamino)-6″-deoxykanamycin A (4c). The compound was synthesized according to the general procedure and obtained as a white solid (47.6 mg, 0.059 mmol,

1 59%). H NMR (500 MHz, D2O) δ 5.57 (d, J = 4.0 Hz, 1H), 5.07 (d, J = 4.0 Hz, 1H), 4.1

- 4.2 (m, 1H), 3.9 - 4.0 (m, 2H), 3.7 – 3.9 (m, 4H), 3.3 – 3.6 (m, 7H) 3.1 – 3.2 (2H), 3.0 –

3.1 (t, J = 8.0 Hz 2H), 2.4 - 2.5 (m, 1H), 1.7 - 1.8 (m, 1H), 1.6 – 1.7 (m, 2H), 1.2 - 1.3 (m,

13 14H), 0.78(t, J = 6.5 Hz, 3H); C NMR (125 MHz, D2O) δ 100.39, 98.52, 83.73, 79.78,

74.19, 71.86, 71.01, 70.75, 68.73, 68.26, 68.05, 67.21, 54.65, 50.14, 48.42, 48.36, 47.72,

40.34, 31.16, 28.65, 28.51, 28.45, 28.35, 28.24, 25.67, 25.38, 22.03, 13.42. ESI/APCI

+ + calcd for C28H58N5O10 [MH] : 624.4184; measured m/e: 624.4181.

6”-(1-Dodecylamino)-6”-deoxykanamycin A (4d). The compound was synthesized

according to the general procedure and obtained as a white solid (54.2 mg, 0.065 mmol,

1 65%). H NMR (500 MHz, D2O) δ 5.52 (d, J = 3.5 Hz, 1H), 5.07 (d, J = 3.5 Hz, 1H), 4.1

- 4.2 (m, 1H), 3.8 - 4.0 (m, 2H), 3.6 – 3.8 (m, 4H), 3.2 – 3.6 (m, 8H), 3.1 – 3.2 (m, 2H),

3.0 – 3.1 (m, 2H), 2.2 - 2.3 (m, 1H), 1.5 - 1.7 (m, 3H), 1.2 - 1.3 (m, 18H), 0.77 (t, J = 7.0

13 Hz, 3H); C NMR (125 MHz, D2O) δ 100.25, 98.78, 84.23, 81.56, 74.38, 72.00, 71.13,

70.84, 68.64, 68.23, 68.14, 67.44, 54.68, 50.40, 48.58, 48.33, 47.75, 40.38, 31.18, 28.77

(3C), 28.69, 28.52, 28.50, 28.42, 25.68, 25.38, 22.03, 13.42. ESI/APCI calcd for 54

+ + C30H62N5O10 [MH] : 652.4497; measured m/e: 652.4487.

6”-(1-Tetradecylamino)-6”-deoxykanamycin A (4e). The compound was synthesized according to the general procedure and obtained as a white solid (40.5 mg, 0.047 mmol,

1 47%). H NMR (500 MHz, D2O) δ 5.56 (d, J = 3.5 Hz, 1H), 5.06 (d, J = 3.5 Hz, 1H), 4.0

- 4.2 (m, 1H), 3.9 - 4.0 (m, 2H), 3.6 – 3.8 (m, 4H), 3.2 – 3.6 (m, 8H), 3.1 – 3.2 (m, 2H),

3.0 – 3.1 (m, 2H), 2.2 - 2.3 (m, 1H), 1.7 - 1.8 (m, 1H), 1.6 – 1.7 (m, 2H), 1.1 - 1.3 (m,

13 22H), 0.78(t, J = 7.0 Hz, 3H); C NMR (125 MHz, D2O) δ 100.43, 98.70, 83.74, 79.93,

74.20, 71.87, 70.99, 70.70, 68.75, 68.29, 68.04, 67.19, 54.64, 50.12, 48.39, 48.34, 47.69,

40.30, 31.19, 28.78 (5C), 28.69, 28.50 (2C), 28.25, 25.68, 25.37, 22.03, 13.41. ESI/APCI

+ + calcd for C32H66N5O10 [MH] : 680.4810; measured m/e: 680.4810.

6”-(1-Hexadecylamino)-6”-deoxykanamycin A (4f). The compound was synthesized

according to the general procedure and obtained as a white solid (17.8 mg, 0.020 mmol,

1 20%). H NMR (500 MHz, CD3OD) δ 5.58 (d, J = 3.5 Hz, 1H), 5.22 (d, J = 3.5 Hz, 1H),

4.2 - 4.3 (m, 1H), 4.0 - 4.1 (m, 1H), 3.7 - 3.9 (m, 4H), 3.4 – 3.7 (m, 7H), 3.0 – 3.3 (m,

6H), 2.4 - 2.5 (m, 1H), 1.7 - 1.9 (m, 3H), 1.3 - 1.4 (m, 26H), 0.92 (t, J = 7.0 Hz, 3H); 13C

NMR (125 MHz, CD3OD) δ 100.29, 97.75, 84.59, 81.29, 73.42, 72.65, 71.87, 71.66,

69.00, 68.76, 68.56, 67.80, 55.12, 50.67, 48.65, 48.39, one carbon peak underneath the

solvent peak, 40.72, 31.66, 29.40 (6C), 29.36, 29.32, 29.21, 29.07, 28.89, 26.28, 25.64,

+ + 22.32, 13.03. ESI/APCI calcd for C34H70N5O10 [MH] : 708.5123, measured m/e:

708.5121.

6″-(1-Hexylmercapto)-6″-deoxykanamycin A (5a). The compound was synthesized according to the general procedure and obtained as a white solid (51.8 mg, 0.071 mmol, 55

1 71%). H NMR (500 MHz, D2O) δ 5.52 (d, J = 4.0 Hz, 1H), 5.01 (d, J = 4.0 Hz, 1H), 3.8

– 4.0 (m, 5H), 3.6 – 3.8 (m, 2H), 3.2 - 3.6 (m, 7H), 3.1 – 3.2 (m, 1H), 2.9 – 3.0 (m, 1H),

2.6 – 2.7 (m, 1H), 2.53 (t, J = 7.5 Hz, 2H) 2.3 - 2.4 (m, 1H), 1.7 - 1.8 (m, 1H), 1.4 - 1.5

13 (m, 2H), 1.1 - 1.3 (m, 6H), 0.76 (t, J = 7.0 Hz, 3H); C NMR (125 MHz, D2O) δ 100.33,

96.70, 84.34, 78.68, 73.19, 72.19 (2C), 70.88, 70.78, 68.59, 68.44, 68.21, 54.82, 49.78,

47.98, 40.38, 32.41, 32.39, 30.63, 28.79, 28.31, 27.64, 21.89, 13.38. ESI/APCI calcd for

+ + C24H49N4O10S [MH] : 585.3164; measured m/e: 585.3171.

6”-(1-Octylmercapto)-6”-deoxykanamycin A (5b). The compound was synthesized

according to the general procedure and obtained as a white solid (40.9 mg, 0.054 mmol,

1 54%). H NMR (500 MHz, D2O) δ 5.54 (d, J = 4.0 Hz, 1H), 5.03 (d, J = 3.5 Hz, 1H), 3.8

– 4.0 (m, 5H), 3.7 – 3.8 (m, 2H), 3.5 - 3.6 (m, 4H), 3.2 – 3.5 (m, 3H), 3.1 – 3.2 (m, 1H),

2.9 – 3.0 (m, 1H), 2.6 – 2.7 (m, 1H), 2.56 (t, J = 7.5 Hz, 2H), 2.4 - 2.5 (m, 1H), 1.8 - 1.9

(m, 1H), 1.5 - 1.6 (m, 2H), 1.1 - 1.4 (m, 10H), 0.78 (t, J = 7.0 Hz, 3H); 13C NMR (125

MHz, D2O) δ 100.46, 96.48, 84.11, 78.03, 72.95, 72.20, 72.11, 70.80, 70.72, 68.65,

68.38, 68.17, 54.80, 49.69, 47.83, 40.35, 32.41, 32.37, 31.10, 28.81, 28.34, 28.28, 27.95,

+ + 27.59, 22.01, 13.42. ESI/APCI Calcd for C26H53N4O10S [MH] : 613.3482; measured

m/e: 613.3486.

6”-(1-Decylmercapto)-6”-deoxykanamycin A (5c). The compound was synthesized

according to the general procedure and obtained as a white solid (35.4 mg, 0.045 mmol,

1 45%). H NMR (500 MHz, D2O) δ 5.52 (d, J = 4.0 Hz, 1H), 5.02 (d, J = 3.5 Hz, 1H), 3.8

– 4.0 (m, 5H), 3.6 – 3.8 (m, 3H), 3.2 - 3.6 (m, 7H), 3.1 – 3.2 (m, 1H), 2.9 – 3.0 (m, 1H),

2.6 – 2.7 (m, 1H), 2.55 (t, J = 7.5 Hz, 2H) 2.3 - 2.4 (m, 1H), 1.7 - 1.8 (m, 1H), 1.5 - 1.6

13 (m, 2H), 1.1 - 1.3 (m, 14H), 0.78 (t, J = 7.0 Hz, 3H); C NMR (125 MHz, D2O) δ 56

100.33, 96.88, 84.49, 79.10, 73.30, 72.16, 72.13, 70.91, 70.78, 68.59, 68.46, 68.23, 54.82,

49.83, 48.02, 40.37, 32.42, 32.39, 31.18, 28.80, 28.70, 28.65 (2C), 28.48, 28.30, 27.93,

+ + 22.04, 13.43. ESI/APCI calcd for C28H57N4O10S [MH] : 641.3795; measured m/e:

641.3789.

6”-(1-Dodecylmercapto)-6”-deoxykanamycin A (5d). The compound was synthesized

according to the general procedure and obtained as a white solid (49.7 mg, 0.061 mmol,

1 61%). H NMR (500 MHz, D2O) δ 5.49 (d, J = 3.5 Hz, 1H), 4.99 (d, J = 3.5 Hz, 1H), 3.8

– 4.0 (m, 4H), 3.2 - 3.7 (m, 10H), 3.0 – 3.1 (m, 1H), 2.9 – 3.0 (m, 1H), 2.6 – 2.7 (m, 1H),

2.52 (t, J = 7.5 Hz, 2H), 2.3 - 2.4 (m, 1H), 1.6 - 1.7 (m, 1H), 1.4 - 1.6 (m, 2H), 1.1 - 1.3

13 (m, 18H), 0.75 (t, J = 7.0 Hz, 3H); C NMR (125 MHz, D2O) δ 100.27, 97.08, 84.65,

79.61, 73.45, 72.17, 72.09, 70.96, 70.80, 68.56, 68.49, 68.27, 54.82, 49.87, 48.10, 40.36,

32.44 (2C), 31.22, 28.82 (4C), 28.76, 28.68, 28.54, 28.34, 27.97, 22.06, 13.44. ESI/APCI

+ + calcd for C30H61N4O10S [MH] : 669.4108; measured m/e: 669.4107.

6”-(1-Tetradecylmercapto)-6”-deoxykanamycin A (5e). The compound was synthesized according to the general procedure and obtained as a white solid (36.2 mg,

1 0.043 mmol, 43%). H NMR (500 MHz, CD3OD) δ 5.61 (d, J = 4.0 Hz, 1H), 5.13 (d, J =

3.5 Hz, 1H), 4.0 – 4.1 (m, 2H), 3.2 – 4.0 (m, 12H), 3.0 – 3.1 (m, 2H), 2.6 – 2.8 (m, 3H),

2.4 - 2.5 (m, 1H), 1.8 – 2.0 (m, 1H), 1.5 - 1.7 (m, 2H), 1.2 - 1.5 (m, 22H), 0.92 (t, J = 7.0

13 Hz, 3H); C NMR (125 MHz, CD3OD) δ 100.41, 95.59, 85.34, 79.29, 73.07 (2C), 72.57,

71.68, 71.64, 68.90, 68.85, 68.63, 55.27, 50.14, one peak underneath the solvent peak,

40.81, 32.77, 32.67, 31.67, 29.49, 29.40 (3C), 29.37 (3C), 29.23, 29.07 (2C), 28.59,

+ + 22.33, 13.03. ESI/APCI Calcd for C32H65N4O10S [MH] : 697.4421; measured m/e:

697.4425. 57

6”-(1-Hexadecylmercapto)-6”-deoxykanamycin A (5f). The compound was synthesized according to the general procedure and obtained as a white solid (27.0 mg,

1 0.031 mmol, 31%). H NMR (500 MHz, CD3OD) δ 5.50 (d, J = 3.5 Hz, 1H), 5.02(d, J =

3.5 Hz, 1H), 3.9 – 4.0 (m, 2H), 3.1 – 4.0 (m, 12H), 2.9 – 3.0 (m, 2H), 2.5 – 2.7 (m, 3H),

2.3 - 2.4 (m, 1H), 1.7 – 1.8 (m, 1H), 1.4 - 1.6 (m, 2H), 1.1 - 1.4 (m, 26H), 0.80 (t, J = 7.0

13 Hz, 3H); C NMR (125 MHz, CD3OD) δ 100.42, 95.56, 85.26, 79.17, 73.06 (2C), 72.57,

71.69, 71.65, 68.88, 68.85, 68.63, 55.28, 50.13, one peak underneath the solvent peak,

40.82, 32.77, 32.67, 31.67, 29.49, 29.39 (6C), 29.35 (2C), 29.07 (3C), 28.59, 22.33,

+ + 13.04. ESI/APCI calcd for C34H69N4O10S [MH] : 725.4734; measured m/e: 725.4730.

6″-(2-(6-hydroxy-3-oxo-3H-xanthen-9-yl))benzamido)-6″-deoxykanamycin A (7). To

a solution of 2 (1.04 gm, 1 equiv, 1.00 mmol) dissolved in 20 mL of anhydrous DMF,

sodium azide (0.214, 3.3 equiv, 3.3 mmol) was added, and the reaction was stirred at 80

oC under nitrogen atmosphere. After being stirred for 48 hrs, solvent was removed under

reduced pressure and the solid residue was washed with water 3x10 mL. The washed

precipitate was dried under vacuum for 24 hours. The dried crude compound was then

dissolved in 10 mL of 1:1 solution of degassed MeOH and AcOH. Catalytic amount of 10

% Pd/C was added, and the reaction mixture was stirred at room temperature for 12 hours

under hydrogen gas environment. The reaction mixture was filtered through Celite and

the solvents were removed. The solid residue was washed with 2x5 mL of 10% sodium

carbonate aqueous solution. The residue was further washed with 2x5 mL of DCM to

obtain compound 6 12. For a solution of compound 6 (0.100 gm, 1 equiv, 0.11 mmol )

dissolved in 10 mL of DMF, NHS activated fluorescein (0.095 gm, 2 equiv, 0.22

mmol)13, was added. The reaction mixture was stirred for 24 hrs at room temperature. 58

Solvent was removed, and the viscous residue was washed with 2x5 mL water and dried

under vacuum. The dried precipitate was treated with a solution of 5 mL of anhydrous

DCM and 1 mL of trifluoro acetic acid, and the reaction mixture was stirred for 6 hrs at

room temperature. After removal of solvents, the viscous residue was loaded to a column

packed with by Amberlite CG50 and eluted with a gradient of solvent (100% water to

20% NH4OH in water). Pure compound obtained after CG50 purification was further

subjected to the ion exchange using anion exchange resin IR410 (Cl- form) to yield the

desired amphiphilic kanamycin as a chloride salt (24.0 mg, 0.025 mmol, 23%). 1H NMR

(500 MHz, D2O) δ 7.8 – 7.9 (m, 1H), 7.5 – 7.6 (m, 2H), 6.9 – 7.0 (m, 1H), 6.6 – 6.7 (m,

2H), 6.55 (d, J = 9.0 Hz, 1H), 6.4 – 6.5 (m, 2H), 6.40 (d, J = 8.5 Hz, 1H), 5.45 (d, J = 3.5

Hz, 1H), 4.60 (d, J = 3.5 Hz, 1H), 3.7 - 3.9 (m, 3H), 3.6 – 3.7 (m, 2H), 3.56 (t, J = 10.0

Hz, 1H), 3.3 - 3.5 (m, 7H), 3.1 – 3.3 (m, 3H), 2.8 - 2.9 (m, 1H), 2.4 – 2.5 (m, 1H), 1.8 -

13 1.9 (m, 1H); C NMR (125 MHz, D2O) δ 171.74, 157.40, 156.99, 153.04, 152.92,

152.77, 134.31, 130.09, 129.61, 129.35, 129.32, 124.24, 122.83, 112.56, 112.08, 111.36,

109.21, 102.65, 102.53, 100.24, 96.26, 84.29, 76.28, 73.96, 72.15, 71.97, 70.67, 70.63,

68.62, 67.30, 66.37, 66.27, 53.68, 49.12, 48.26, 41.05, 40.05, 27.47. ESI/APCI calcd for

+ + C38H48N5O14 [MH] : 798.3198; measured m/e: 798.3165.

Procedure for the Antifungal Activity. Minimum inhibitory concentrations of the compounds were evaluated by two-fold dilution method following standard protocol [14,

15]. All the fungal strains were grown in RPMI medium supplement with 0.165 M MOPS buffer (pH = 7) at 28oC with gentle shaking for 48 hours. Compounds were dissolved in water to make stock solution of 10 mg/mL. The compounds from stock were diluted in 96 well plate using growth medium to the final concentration ranging from 512 – 0.25 59

µg/mL. 50 µL of fungi with recommended confluence from the standard protocol was

added to each well containing 50 µL of diluted compounds and incubated for 24 to 36 h.

The minimum concentration of compound that inhibits fungal growth to visible clearance

is reported as the MIC value.

Procedure for the Antibacterial Activity. Bacterial strains were grown in Lysogeny

broth (LB) at 35oC. MIC of compounds against was also determined by two-fold dilution method. The compounds were diluted in a 96-well plate using water to the final concentration from 512 – 0.25 µg/mL. 50 µL of bacteria growth with adjusted confluence

(OD600 = 0.08 – 0.1) was added to each well of the 96-well plate with 50 µL of diluted

compounds. The plate was incubated for 18 hrs at 35oC and read visually. The minimum

concentration of compound that inhibited the bacterial growth to visible clearance is

reported as the MIC value.

Procedure for the Evaluation of Cytotoxicity Towards Human Cell. Cytotoxicity of

the compounds was evaluated by colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-

2,5-diphenyltetrazolium bromide (MTT) dye. The compounds’ cytotoxicity towards

human cells was evaluated against the HeLa cell, a cervical human cell. Cells were grown

in DMEM medium (Corning) supplemented with 10% fetal bovine serum and 100 U/mL

o each of penicillin and streptomycin under 5% CO2 in humid environment at 37 C. 200 µL

of cell suspension was added to each well of a 96-well plate from 25,000 cells/mL stock

and incubated for 24 hrs. The medium was removed, and the compounds were? diluted in

growth medium from 100 µg/mL - 0.1 µg/mL with ten-fold dilution along with just

medium was added to the cells. After 24 h of incubation, 20 µL of MTT dye (5 mg/mL in

PBS) was added and incubated for 5 more hrs. The medium was removed, and the 60

reduced formazan dye was dissolved in 200 µL of DMSO. Absorption of formazan

solution was measured at 570 nm with 650 nm as the background absorption. The

experiment was done in triplicate of triplicate. During the analysis, absorption from the

control was converted to 100% cell viability and the cell viability at different

concentration of the compounds were calculated relative to the control.

Procedure for the Imaging of Fungi Incubated with Compound 7. Fungi were grown

in the PDB medium with gentle shaking at 28oC for 48 hrs. 1 mL of fungal growth was transferred to the centrifuge tube and spun down at 10,000 rpm for 2 minutes in a Fisher

Scientific accuSpin MicroCentrifuge at room temperature. Cell pallets were washed one more time using the same volume of the water. The fungal cells were then incubated with

1 mL of 32 µg/mL of compound 7 for 5 or 60 minutes. The final confluence of fungal cells was maintained to 1x107 cells/mL during the incubation with compound 7. After the incubation with compound 7, fungal cells were washed with the same volume of water by centrifugation and resuspended in 1 mL of water. Images of fungi were taken using an

Olympus IX71 microscope using 100X oil immersion objective under phase contrast and green channel.

Procedure for the Imaging of Bacteria Incubated with Compound 7. Bacteria were

grown in LB medium at 37oC for 18 hours. 1 mL of bacteria in each centrifuge tube was

spun down at 10,000 rpm for 2 minutes in a Fisher Scientific accuSpin Micro at room

temperature. Bacterial cells were washed with water twice using same volume of water

by centrifugation. Bacterial cells were then treated with 1 mL of 32 µg/mL of compound

7. After 5 or 60 minutes of incubation, cells were washed with water twice. After

washing, cells were resuspended in 100 µL of water. Images were taken in Olympus IX71 61

microscope using 100X oil immersion objective under phase contrast and green channel.

2.7 References.

[1] Y.P. Subedi, M.N. AlFindee, J.Y. Takemoto, C.-W.T. Chang, Antifungal

amphiphilic kanamycins: new life for an old drug, MedChemComm, 9 (2018) 909-

919. DOI: 10.1039/C8MD00155C.

[2] S.K. Shrestha, M.Y. Fosso, K.D. Green, S. Garneau-Tsodikova, Amphiphilic

tobramycin analogues as antibacterial and antifungal agents, Antimicrob. Agents

Chemother., 59 (2015) 4861-4869. DOI: 10.1128/AAC.00229-15.

[3] M.Y. Fosso, S.K. Shrestha, K.D. Green, S. Garneau-Tsodikova, Synthesis and

bioactivities of kanamycin B-derived cationic amphiphiles, J. Med. Chem., 58

(2015) 9124-9132. DOI: org/10.1021/acs.jmedchem.5b01375.

[4] R. Dhondikubeer, S. Bera, G.G. Zhanel, F. Schweizer, Antibacterial activity of

amphiphilic tobramycin, J. Antibiot., 65 (2012) 495. DOI: 10.1038/ja.2012.59

[5] I.M. Herzog, K.D. Green, Y. Berkov‐Zrihen, M. Feldman, R.R. Vidavski, A.

Eldar‐Boock, R. Satchi‐Fainaro, A. Eldar, S. Garneau‐Tsodikova, M. Fridman, 6′′‐

Thioether Tobramycin Analogues: Towards Selective Targeting of Bacterial

Membranes, Angew. Chem. Int., 51 (2012) 5652-5656. DOI:

10.1002/anie.201200761.

[6] S.K. Shrestha, C.-W.T. Chang, N. Meissner, J. Oblad, J.P. Shrestha, K.N. Sorensen,

M.M. Grilley, J.Y. Takemoto, Antifungal amphiphilic aminoglycoside K20:

bioactivities and mechanism of action, Front. Microbiol., 5 (2014) 671. DOI:

10.3389/fmicb.2014.00671.

[7] A. Van Schepdael, J. Delcourt, M. Mulier, R. Busson, L. Verbist, H. Vanderhaeghe, 62

M.-P. Mingeot-Leclercq, P.M. Tulkens, P. Claes, New derivatives of kanamycin B

obtained by modifications and substitutions in position 6''. 1. Synthesis and

microbiological evaluation, J. Med. Chem., 34 (1991) 1468-1475. DOI:

10.1021/jm00108a035.

[8] M.D. Disney, O.J. Barrett, An aminoglycoside microarray platform for directly

monitoring and studying antibiotic resistance, Biochemistry, 46 (2007) 11223-

11230. DOI: 10.1021/bi701071h.

[9] K.B. Steinbuch, R.I. Benhamou, L. Levin, R. Stein, M. Fridman, Increased degree

of unsaturation in the lipid of antifungal cationic amphiphiles facilitates selective

fungal cell disruption, ACS Infect. Dis., 4 (2018) 825-836. DOI:

org/10.1021/acsinfecdis.7b00272.

[10] S.F. Yeo, B. Wong, Current status of nonculture methods for diagnosis of invasive

fungal infections, Clin. Microbiol. Rev., 15 (2002) 465-484. DOI:

10.1128/cmr.15.3.465-484.2002.

[11] Y.P. Subedi, P. Roberts, M. Grilley, J.Y. Takemoto, C.-W.T. Chang, Development

of fungal selective amphiphilic kanamycin: Cost-effective synthesis and use of

fluorescent analogs for mode of action investigation, ACS Infect. Dis., 5 (2019)

473-483. DOI: org/10.1021/acsinfecdis.8b00327.

[12] R.J. Fair, M.E. Hensler, W. Thienphrapa, Q.N. Dam, V. Nizet, Y. Tor, Selectively

guanidinylated aminoglycosides as antibiotics, Chem. Med. Chem., 7 (2012) 1237-

1244. DOI: 10.1002/cmdc.201200150.

[13] J. Gao, P. Wang, R.W. Giese, Xanthamide fluorescent dyes, Anal. Chem., 74 (2002)

6397-6401. DOI: org/10.1021/ac020368+. 63

[14] CLSI, Reference method for broth dilution antifungal susceptibility testing of

yeasts; approved standard—second edition—document M27-A2, in, CLSI, Wayne,

PA, USA, 2002.

[15] CLSI, Reference method for broth dilution antifungal susceptibility testing of

filamentous fungi; approved standard—second edition—document M38-A2, in,

CLSI, Wayne, PA, USA, 2008.

CHAPTER 3

DEVELOPMENT OF FUNGAL SELECTIVE AMPHIPHILIC KANAMYCIN: COST

EFFECTIVE SYNTHESIS AND USE OF FLUORESCENT ANALOGS

FOR MODE OF ACTION INVESTIGATION a

3.1 Abstract

Amphiphilic aminoglycosides have attracted interest due to their novel

antifungal activities. A crucial but often neglected factor for drug development in

academia is cost of production. Herein is reported a one-step, inexpensive synthesis

of amphiphilic kanamycins constituted with only natural components. The synthetic

methodology aslo enabled the preparation of series fluorescent amphiphilic aryl

kanamycins for direct structure-activity mode of action studies. The lead compounds

showed prominent antifungal activity against a panel of fungi, including Fusarium

graminearum, Cryptococcus neoformans, Aspergillys fumigatus, Candida auris and

various Candida sp., and significant antibacterial activities. With the fluorescence

based whole cell assays, the aryl amphiphilic kanamycins was observed to

permeabilize fungal surface membranes at faster rates than bacterial surface

membranes.

aAdapted with permission from (Subedi, Y. P.; Roberts, P.; Grilley, M.; Takemoto, J. Y.; Chang, C.-W. T. “Development of fungal selective amphiphilic kanamycin: Cost- effective synthesis and use of fluorescent analogs for mode of action investigation” ACS Infect. Dis., 2019, 5, 473-483). Copyright © 2019, American Chemical Society 65

Also, the antifungal action of the amphiphilic kanamycins was observed to occur in a biphasic mode with an initial fast phase correlated with rapid membrane permeabilization at subminimal inhibitory concentrations and a slower phase membrane permeabilization that elevates the reactive oxygen species production leading to cell death. Inactive hydrophobic amphiphilic kanamycins displayed no membrane permeabilization. The results offer cost- effective methods for producing amphiphilic kanamycins and reveal insights into how nonfungal specific amphiphilic kanamycins can be employed for fungal specific diagnostic and therapeutic applications. 66

3.2 Introduction

Aminoglycoside antibiotics, once effective against infectious bacterial infections,

are plagued with the rampage of antibiotic resistant bacteria.1 To counteract this problem,

modifications of aminoglycoside antibiotics have been studied extensively with the goal

of reviving antibacterial activity.2-5 Recently, the discovery of amphiphilic

aminoglycosides provides a new strategy and approach in the fight against resistant

bacteria.6 Unlike traditional aminoglycosides, amphiphilic aminoglycosides have been

shown to increase the membrane permeability for both bacteria and fungi.7-14 The

antifungal activity of amphiphilic kanamycin (AK) is of particular interest as it represents

a new strategy of re-purposing and reviving the use of an old drug.15-17

To provide cost effective antimicrobials, our group has focused on low cost

syntheses of AK for uses in green agriculture and human medicine (Figure 1). The lead

compound, K20, displayed effectiveness in controlling Fusarium head blight in wheat

field trials.18,19 In addition, combinations of K20 and half-label rates of commonly

employed agrofungicides significantly lowered deoxynivalenol (DON) mycotoxin levels

in harvested grain. Nevertheless, two shortcomings are associated with K20. First, the cost of production of K20 is not compatible with agrofungicides currently used in the market.20 Second, K20 contains a non-natural structural scaffold that makes it difficult to

be classified as a natural or organic fungicide. Also, questions still linger regarding the

antifungal mode of action of the AKs. Several reports have shown that AKs increase the

permeability of fungal membranes.6-12 However, it is unclear whether this is the sole

mode of action (MOA) against fungi. Several studies have reported AKs that are active

against both fungi and bacteria (non-fungal specific)6-10 while K20 and FG08 are active 67 only against fungi. Therefore, it is of interest to determine the factors that cause AKs to be fungal or non-fungal specific agents.

NH NH2 6' 2 4' I I 3 O HO O R 2 R 1' HO 1' NH II NH2 II 3' R1 2 R1 O NH O NH HO 2 HO 2 O O III amphiphilic kanamycin III 2 5" 6" R kanamycin class 1" OH 1" HO O HO O 3 antibiotics OH OR 4 4" H2N R 2 3 R1 R2 R3 R1 R R R4 Kananycin A OH OH OH K20 OH OS(O)2C8H17 H NH2 FG08 NH C H OH Kananycin B OH NH2 OH 2 H 8 17 Dibekacin NH2 H H Tobramycin NH2 H OH

Figure 3-1. Structures of selected kanamycin class antibiotics and Aks

3.2 Results and Discussion

Cost-Eﬀective Synthesis of Amphiphilic Kanamycins

To address the cost and natural product issues mentioned above about AKs, we explored the possibility of AK synthesis in a low cost one-step modification of kanamycin. We selected kanamycin sulfate and fatty acids as the low cost starting materials and attempted one-step regioselective acylation of the amino groups on kanamycin A. The fatty acids were converted into the corresponding esters of N- hydroxysuccinimide with modification of a previously reported method.21 Slow addition of these esters to a solution of kanamycin A afforded the desired AKs (Scheme 1).

Modest to excellent yields were achieved for the one-step regioselective acylation. Based 68 on the chemicals needed, the cost of production of these AKs is about 1/10 of that for

K20. Since both fatty acids and kanamycin are natural products, the newly synthesized

AKs are anticipated to be classified as natural, thus meeting our goals of price competitiveness and natural and green products. The same synthetic method was used to synthesize 3f using tobramycin as the starting material. Three derivatives, 2h-j with aryl carboxyl group were also synthesized using the same synthetic methods for potential

MOA with direct visualization of time-based uptake into the fungi, bacteria and mammalian cell. 69

Scheme 3-1. Synthesis of amphiphilic aminoglycosides

Assay of Antifungal and Antibacterial Activities

The newly synthesized AKs were tested for their antibacterial and antifungal activities, and cytotoxicity towards mammalian cells. For antifungal activity, a collection of significant human and plant fungal pathogens were used, including Aspergillus flavus

(human and plant pathogen), Fusarium graminearum (plant pathogen), Candida albicans 70

64124 (human pathogen, azole-resistant), C. albicans MYA2876 (human pathogen, azole

susceptible), Cryptococcus neoformans H99 (human pathogen), and Rhodotorula

pilimanae, using voriconazole as the controls. From the minimum inhibitory

concentration (MICs), the compounds with linear alkyl chain showed a clear SAR: the

antifungal activity increases as the chain length increases (Table 1). The antifungal

activity is broad spectrum among various strains, which becomes optimal with the

attachment of hexadecyl (C16) (2f and 3f) and octadecyl (C18) (2g) groups. By comparing the outcomes of 2f and 3f, it appears that the core kanamycin is not a significant factor for creating antifungal activity. For AKs with aryl groups, compound 2i

shows the best antifungal activity followed by 2j whereas 2h is inactive.

Table 3-1. MICs of AKs against fungi[a]

Compound A[b] B[b] C[b] D[b] E[b] F[b]

2a ND[c] ≥256 ≥256 ≥256 ≥256 ≥256

2b ND ≥256 ≥256 ≥256 ≥256 ≥256

2c ND ≥256 ≥256 ≥256 128 128

2d 256 16 32 32 8 16

2e 256 8 16 16 4 4

2f 32 4 8 8 4 4

2g 16 4 8 8 4 4

2h ND ≥256 ≥256 ≥256 128 256

2i ≥256 16 16 16 16 16

2j ≥256 32 64 128 32 16

3f ND 2 4 4 2 4

K20 32 8 16 16 8 4

FG08 ND 7.8 ND ND ND 7.8

Kanamycin A ND >128 ≥256 ≥256 ≥256 ND

Voriconazole 1 32 ≥256 0.125 0.125 8

[a] Unit: µg/mL; [b] A: A. flavus, B: F. graminearium B4-5A, C: C. albicans 64124,

D: C. albicans MYA2876, E: C. neoformans H99, F: R. pilimanae; [c] ND: Not determined.

Since the addition of linear alkyl chain showed superior activities, we selected several of these newly synthesized versions of kanamycin derivatives and investigated their activity against clinically emerging and significant Candida species, including C. albicans B-311, C. rugosa 95-967, C. parapsolis Cas08-0490 (azole resistant), C. tropicalis 95-41. C. albicans B-311 has been reported to exhibit biotin-independent growth.22 C. rugosa is an emerging human fungal pathogen in some regions with decreased susceptibility to fluconazole, a commonly used antifungal agent.23 C. parapsolis is often the second or most commonly isolated Candida species from blood cultures.24 C. tropicalis is the second most virulent Candida species found in skin, gastrointestinal tract, and in female genitourinary tract that is capable of producing biofilm.25 The MICs values show that the leads compounds are still 2f, 2g and 3f.

Table 3-2. Minimum inhibitory concentration of AKs against Candida sp.[a]

Compound A[b] B[b] C[b] D[b]

2c ≥256 ≥256 ≥256 ≥256

2d 128 64 128 128

2e 16 16 32 32

2f 8 8 16 16

2g 8 8 8 16

2i 64 64 128 128

3f 8 8 8 16

K20 16 16 32 32

[a] Unit: µg/mL; [b] A: C. albicans B-311, [B]: C. rugosa 95-967, C: C. parapsolis Cas08-0490 (azole resistant), D: C. tropicalis 95-41.

The leads 2f and 2g are synthesized on a larger scale and tested for the more

human pathogens (Table 3-3). The results show that 2g have excellent activity towards

susceptible and fluconazole resistance Candia auris with MIC values from 0.5 to 1

µg/mL. The tested compounds are also active towards Aspergillus fumigatus.

Table 3-3. MICs of 2f and 2g against human fungal pathogens[a]

Compound A[b] B[b] C[b] D[b] E[b] F[b]

2f 8 4 8 4 4 8

2g 8 4 1 0.5 0.5 4

Fluconazole 16 >64 ≥64 ≥64 2 ND

Voriconazole ND ND ND ND ND 1

[a] Unit: µg/mL; [b] A: C. krusei, B: C. albicans CA3, C: C. auris DI17-46, D: C.

auris DI17-47 E: C. auris DI17-48, F: Aspergillus fumigatus AF1; [c] ND: Not

determined.

For the test of antibacterial activity, we selected representative strains of bacteria,

including Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), S.

aureus (ATCC 33591, MRSA), and S. aureus (ATCC 43300, MRSA). The AKs with

linear alkyl chain showed a similar SAR as their antifungal activity: the antibacterial activity increases as the chain length increases with the most active one bearing hexadecyl or octadecyl groups (Table 3-4). An interesting difference has been noted: the

AKs with octyl groups attached to ring III of kanamycin at the O-6″ or O-4″ positions, such as K20 and FG0811,18 are considered inactive (MICs ≥256 µg/mL) against bacteria

while exerting noticeable antifungal activity, i.e. fungal specific. In contrast, the 74

attachment of an octyl group at the N-6′ position of kanamycin (2b) does not manifest either antifungal or antibacterial activity. Comparing the antifungal and antibacterial activities, the bioactivity profile of the newly synthesized AKs appears to resemble those with alkylthio groups at O-6″ position.12 The combined SAR study results reported herein and by others indicate that long linear alkyl groups (C14-C18) is advantageous in exerting antifungal and antibacterial activity, i.e. non-fungal specific. However, these

AKs on average show better antifungal activity.

Table 3-4. MICs of AKs against bacterial strains[a]

Compound A[b] B[b] C[b] D[b]

2a ≥256 ≥256 ≥256 ≥256

2b ≥256 ≥256 ≥256 ≥256

2c ≥256 ≥256 ≥256 ≥256

2d 32 32 128 128

2e 64 16 32 32

2f 32 16 16 16

2g 64 32 32 32

2h ≥256 ≥256 ≥256 ≥256

2i 128 32 128 32

2j 128 128 ≥256 ≥256

3f 32 16 32 32

K20 256 128 >256 >256

FG08 ≥256 ≥256 NDc ND

[a] Unit: µg/mL; [b] A: E. coli (ATCC 25922), B: S. aureus (ATCC 25923),

C: S. aureus (ATCC 33591) MRSA, D: S. aureus (ATCC 43300) MRSA; [c]

ND: Not determined.

Membrane Permeabilization and Cytotoxicity Study

It has been reported that non-fungal specific AKs cause increases in fungal and bacterial membrane permeabilities.12,14 Many of these studies were accomplished using fungi treated with AKs with long incubation time (hrs). Thus, we were interested in exploring whether non-fungal specific AKs can behave like K20 or FG08, fungal specific AKs, with fast membrane permeabilization properties. We used fluorogenic sytox green dye to investigate the kinetic membrane permeabilization effect of newly synthesized AKs using C. albicans MYA2876. The fungi were treated with two AKs, 2b and 2g to observe time-dependent membrane permeabilization. The active 2g caused a drastic fluorescence increase within minutes. while almost no fluorescence increase was observed for the cells treated with 2b even after 2 hrs compared to control (Figure 2). The inability of the 2b to permeabilize the fungal membrane is consistent with its lack of antifungal activity. This outcome confirms that 2g causes membrane permeabilization consistent with the fast-acting nature of K20 or FG08 and other membrane permeabilizing agents.

Figure 3-2. Kinetic membrane permeabilization study of the AKs. (A): Time-dependent measurement of sytox green fluorescence in relative fluorescence unit (RFU); ((B) and

(C): images of C. albicans MYA2876 treated with 2b for 60 min.; (D) and (E): images of C. albicans MYA2876 treated with 2g for 60 min.

26 The cytotoxicity (IC50) of AKs were also determined using HeLa cells. From the

IC50 values, the cytotoxicity increases as the chain-length of the AKs with linear alkyl group at N-6′ position increases where as the typical aminoglycosides in the natural form are reported non-toxic up to the 100 µg/mL concentration.27, 28 The result from newly synthesized AKs is consistent with the feature of non-selective membrane targeting antiseptic agents. However, the cytotoxicity for the lead AKs, 2f and 2g (IC50 of 64.6 and

63.0 µg/mL, respectively) is still lower than their antifungal and antibacterial activities.

The AK’s with aryl (fluorescent) groups and K20 and FG08 are considered much less toxic (IC50 >100 µg/mL). 78

Uptake Study of Amphiphilic Kanamycin Using Fluorescent Analog

To directly observe cell uptake and interaction of the new AKs during membrane

permeabilization, AKs (2h-j) with aryl group were synthesized. However, 2h was found to be non-fluorescent while 2i and 2j show strong fluorescence.29 Thus, our fluorescence-

based investigation was directed to the use of 2i and 2j. A recent report showed that AKs

incorporated with fluorophores “rapidly accumulated in the cytosol and led to structural

changes in proteins and DNA.”30 In the current study, we chose to use hydrophobic

fluorophores instead of an alkyl chain/fluorophore combination so that the overall

structures of 2i and 2j would more closely resemble the amphipathic structural features of

the AKs.

First, 2j was incubated at 32 μg/mL, which is one, ¼ and ½ of the MICs against

F. graminearum, C. albicans MYA2876 and C. albicans 64124, respectively (Figure 3-

3). Interestingly, fluorescence from 2j closely resembled previously reported images30

and was observed to physically associate with cells of F. graminearum and the two C.

albicans strains within 5 minutes of initial exposure. Therefore, it is likely that AKs not

only caused fast membrane permeabilization but also quickly interacted with cells at

concentrations lower than the corresponding MICs. Incubation for 5 or 60 minutes

revealed preferential localization of 2j on or in the cells observed as subcellular areas of higher fluorescence intensities. For 2i, F. graminearum, C. albicans MYA2876 and C.

ablicans 64124 were treated with 16 µg/mL, corresponding to their respective MICs

(Figure 3-4). Again, images show a fast (within 5 minutes) association with cells.

Figure 3-3. Images of fungi treated with fluorescent 2j. (A) and (B): F. graminearum treated for 5 min.; (C) and (D): F. graminearum treated for 60 min.; (E) and (F): C. albicans MYA2876 treated for 5 min.; (G) and (H): C. albicans MYA2876 treated for

60 min.; (I) and (J): C. albicans 64124 treated for 5 min.; (K) and (L): C. albicans

64124 treated for 60 min.

Figure 3-4. Images of fungi treated with fluorescent 2i. (A) and (B): F. graminearum

treated for 5 min.; (C) and (D): F. graminearum treated for 60 min.; (E) and (F): C.

albicans MYA2876 treated for 5 min.; (G) and (H): C. albicans MYA2876 treated

for 60 min.; (I) and (J): C. albicans 64124 treated for 5 min.; (K) and (L): C. albicans

64124 treated for 60 min.

Fluorescent compounds 2i and 2j containing aryl group were also investigated for their kinetic membrane permeabilization property using a second fluorescent dye, propidium iodide (PI) (red fluorescence) (Figure 6). C. albicans 64124 was treated with

2i and 2j at their corresponding MICs (16 µg/mL for 2i and 64 µg/mL for 2j). Again, a 81

fast increase of fluorescence from PI was observed when cells were treated with 2i and 2j

(Figure 5A). The images were taken after 2 hrs of incubation and after observed in three

different fluorescent channels, blue, green and red reflecting the presence of 2i, 2j, and

PI, respectively (Figure 5B-G) along with bright field image. When cells treated with 2i

and 2j, fluorescence from all of these compounds can be seen. In contrast, no

fluorescence increase was observed when cells were treated with PI alone.31 Interestingly,

the profile of relative fluorescence unit (RFU) for 2i resembles the one from 2g while the

RFU of 2j appears to have two or more phases. The difference may be attributed to the molecular designs: 2i resembles more closely to 2g as an AK while 2j has polar

functional groups (C=O and OH) on the hydrophobic fluorophore. It is possible that such

a structural difference changes the MOA events and kinetics.

Figure 3-5. Kinetic membrane permeabilization study of 2i and 2j with PI. A): time-

dependent measurement of fluorescence in relative fluorescence unit (RFU); (B) - (D):

images of C. albicans strain 64124 treated with 2i and PI for 2 hrs under bright field,

blue channel and red channel.; (E) – (G): images of C. albicans strain 64124 treated

with 2j and PI for 2 hrs under bright field, green channel and red channel.

To investigate the membrane selectivity of AKs, bacteria (E. coli and S. aureus)

were incubated with 2j at MIC (128 μg/mL) and 1/4 MIC (32 μg/mL) of both bacteria

and observed the fluorescence after 5 and 60 min. of incubation. The images showed that

2j can still cause membrane permeabilization of bacteria but higher concentrations (at

least at MIC) and longer incubation time are needed.31 Human cells (HeLa, cervical

cancer cells) were incubated with 2j at 32 µg/mL. No fluorescence was noted after incubation for 5 min and 60 minutes.31 These results offer useful information on the rate

of membrane permeabilization caused by AKs as in the order of

fungi>bacteria>>mammalian cells. This finding, which cannot be revealed by traditional 83

assays such as MIC determination, implies that it is possible to employ AKs for detection

or as a diagnostic tool selective toward fungi even though the AKs reported herein and by

others show activity against both fungi and bacteria.

Evaluation of Reactive Oxygen Species

Production of cellular reactive oxygen species (ROS) is a common mode of action

for antibiotics of various classes.32 Aminoglycosides are reported as the ROS generator in

bacteria and mammalian cells.28, 33, 34 Therefore, we decided to examine the production of

cellular ROS in fungi treated with the AKs. Four members of the newly synthesized AKs

and K20 were investigated using 2′,7′-dichlorofluorescein diacetate (DCF-DA) dye.

Compound 4 (1,1'-(hexane-1,6-diyl)bis(3-decyl-4,9-dioxo-4,9-dihydro-1H-naphtho[2,3-

d][1,2,3]triazol-3-ium triflate), a cationic anthraquinone analog developed in our group

and known to trigger the production of ROS, was used as the positive control.35, 36 DCF-

DA dye is non-fluorescence itself and turn in to fluorescnet 2',7'-dichlorofluorescein

(DCF) once hydrolyzed by the esterage and then oxidised by the ROS species. Incubation

of these selected compounds with C. albicans MYA2876, reveals that all of the active

AKs lead to elevated levels of cellular ROS comapared to the control having cells only

(Figure 6). The inactive one, 2b, has ROS level similar to the blank control. From the

ROS production study, it is reasonable to propose that AKs exert their biological activity

by membrane perturbation and then enter the cells causing ROS production, which leads

to cascades of cellular oxidative damage and eventual cell death. Particular membrane

lipids such as sphingolipids with 4-hydroxylated sphinganine backbones and

inositolphosphate-containing head groups that are unique to fungi may play a crucial role

in the fungi specific activity of AKs.13 Therefore, the reason that most of the reported 84

AKs display lower cytotoxicity is likely due to the fact that these AKs simply cannot

penetrate the mammalian plasma membrane as quickly as they do with bacterial or fungal

membranes.

Figure 3-6. Cellular ROS production

3.4 Conclusion

We have developed a one-step cost-effective synthesis of a library AKs from kanamycin. Compared to the common agrofungicides used in agriculture, the cost of production for the lead compounds consisting of only natural components is competitive or even lower. The revealed SAR indicates that long linear alkyl chain (C14-C18) is crucial to generate significant antifungal and antibacterial activities. The analysis of antifungal activity provides a characteristic profile for non-selective membrane-targeting

agents. The lead AKs also exhibit minimal cytotoxicity against mammalian cells. From 85

the kinetic membrane permeabilization and fluorescence imaging studies, we discover

that, even for non-fungal specific AKs, it is possible to achieve fungi selective membrane

permeabilization by controlling the time of incubation and concentration of AKs, and

thus avoid inducing bacterial resistance. Once the integrity of the microbial membrane is

compromised, the fast acting AKs can enter the calls, lead to the production of ROS and cell death in disrupting the functions of multiple cellular targets. The designs of fluorescent AKs that closely resemble the structural features of non-fluorescent AKs provides direct visualization of AKs in action and represents a sensitive and selective diagnostic tool for fungi.

3.5 Experimental Section

Material and Methods. Chemicals were purchased from commercial sources and used without further purification. Fluorescence profile of the compounds was measured in

Spectro fluorophotometer (Shimadzu, RF-5301PC) and fluorescence intensity for the membrane permeabilization study was measured in Cytation 5 imaging reader.

Fluorescence images of the fungi were taken in Olympus IX innervated fluorescence microscope and Cytation 5 imaging reader. 1H-NMR and 13C-NMR of the compounds

were recorded in Bruker AvanceIII HD Ascend-500 at 283 K temperature.

General Procedure for the Synthesis of 2a-2c. To 0.582 gm (1 equiv., 1 mmol) of kanamycin sulfate was dissolved in the 10 mL of water 2 equiv. of potassium carbonate was added and stirred for 15 minutes. Then 1.5 equiv., 1.5 mmol of NHS-acyl ester dissolved in 10 mL of MeOH was added in 4 portions with one-hour interval. After 24 hours of reaction, solvent was removed by air blow and the compound was purified by 86

the column chromatography using Methanol to 10% NH4OH in MeOH. Pure compounds in neutral form were acidified with 5% acetic acid and then air blow to dryness. The compound with acetate counter ion was pass through the IR410 ion exchange resin (in Cl- form) to get the compound with chloride counter anions.

General procedure for the synthesis of 2d – 2j. 0.582 gm (1 equiv., 1mmol) of kanamycin and 3 equiv. of potassium carbonate was dissolved in the 10 mL of water; then 2 equiv. of NHS-acyl ester dissolved in 10 mL of DMF was added in 4 portions with one-hour interval. After 48 hours of reaction, solvent was removed by the air blow and the compound was purified by column chromatography using MeOH to 10% NH4OH in

MeOH. These compounds were also converted to the cationic form and chloride similar to the compounds 2a-2c.

Large Scale Synthesis of 2f and 2g. 5.82 gm (1 equiv., 10 mmol) of kanamycin sulfate and 2.76 gm (2 equiv., 20 mmol) of potassium carbonate dissolved in 50 mL of water and stirred vigourously for 15 minutes at room temperature. 50 mL DMF was added slowly in to the reaction flask, over the period of 15 minutes. Then 2 equivalent of NHS-ester of fatty acid dissolved in 50 mL of DMF was slowly added in four portion in the interval of an hour. After 48 hours of reaction, solvent was removed by air blow. White solid obtained was extracted with 3x300 mL of 5% acetic acid in water. The solvent in the extract was removed under reduced pressure and the solid obtained was subjected to iion exchange to obtain final compounds in the chloride form.

6′-N-Hexanoylkanamycin A (2a). This compound was synthesized as white solid by the reaction of Kanamycin A sulfate with NHS-hexyl ester, synthesized as before37, 87

1 following the general procedure. H NMR (500 MHz, D2O) δ 5.38 (d, J = 4.0 Hz, 1H),

5.04 (d, J = 3.5 Hz, 1H), 3.8-3.9 (m, 3H), 3.6 – 3.8 (m, 7H), 3.4 - 3.6 (m, 6H), 3.24 (t, J =

9.0 Hz, 1H), 2.4 - 2.5 (m, 1H), 2.20 (t, J = 7.0 Hz, 2H), 1.8 - 1.9 (m, 1H), 1.4 - 1.6 (m,

2H), 1.1 - 1.3 (m, 4H), 0.79 (t, J = 7.0 Hz, 3H); 13C NMR (125 MHz, D2O) δ 178.04,

100.44, 97.94, 83.55, 79.25, 72.92, 72.73, 72.12, 71.28, 71.10, 70.10, 68.15, 65.34,59.74,

54.99, 49.70, 48.23, 39.10, 35.73, 30.47, 27.68, 25.12, 21.65, 13.24. ESI/APCI Calcd for

+ + C24H47N4O12 [MH] : 583.3190; Measured m/e:583.3192.

6′-N-Octanoylkanamycin A (2b). This compound was synthesized as white solid by the

reaction of Kanamycin A sulfate with NHS-octyl ester, synthesized as before38, following

the general procedure.1H NMR (500 MHz, D2O) δ 5.38 (d, J = 4.0 Hz, 1H), 5.04 (d, J =

3.5 Hz, 1H), 3.8-3.9 (m, 3H), 3.6 – 3.8 (m, 7H), 3.4 - 3.6 (m, 6H), 3.24 (t, J = 9.5 Hz,

1H), 2.4 - 2.5 (m, 1H), 2.20 (t, J = 7.5 Hz, 2H), 1.8 - 1.9 (m, 1H), 1.4 - 1.6 (m, 2H), 1.1 -

1.3 (m, 8H), 0.78 (t, J = 7.0 Hz, 3H); 13C NMR (125 MHz, D2O) δ 178.03, 100.45, 98.03,

83.62, 79.44, 73.00, 72.72, 72.15, 71.27, 71.14, 70.09, 68.16, 65.33,59.72, 54.99, 49.73,

48.26, 39.06, 35.76, 30.98, 28.11, 28.04, 27.85, 25.41, 21.94, 13.37. ESI/APCI Calcd for

+ + C26H51N4O12 [MH] : 611.3503; Measured m/e: 611.3484.

6′-N-Decanoylkanamycin A (2c). This compound was synthesized as white solid by the

reaction of Kanamycin A sulfate with NHS-decyl ester, synthesized as before38, following the general procedure. 1H NMR (500 MHz, D2O) δ 5.38 (d, J = 4.0 Hz, 1H),

5.04 (d, J = 3.5 Hz, 1H), 3.8-3.9 (m, 3H), 3.6 – 3.8 (m, 7H), 3.4 - 3.6 (m, 6H), 3.24 (t, J =

9.5 Hz, 1H), 2.4 - 2.5 (m, 1H), 2.20 (t, J = 7.5 Hz, 2H), 1.8 - 1.9 (m, 1H), 1.4 - 1.6 (m,

13 2H), 1.1 - 1.3 (m, 12H), 0.78 (t, J = 7.0 Hz, 3H); C NMR (125 MHz, D2O) δ 178.04,

100.45, 98.07, 83.62, 79.47, 73.02, 72.72, 72.16, 71.26, 71.14, 70.09, 68.16, 65.32,59.71, 88

54.99, 49.73, 48.26, 39.07, 35.75, 31.12, 28.60, 28.39, 28.34, 28.14, 27.86, 25.40, 22.04,

+ + 13.41. ESI/APCI Calcd for C28H55N4O12 [MH] : 639.3816; Measured m/e: 639.3788.

6′-N-Dodecanoylkanamycin A (2d). This compound was synthesized as white solid by

the reaction of Kanamycin A sulfate with NHS-dodecyl ester, synthesized as before38,

1 following the general procedure. H NMR (500 MHz, CD3OD) δ 5.37 (d, J = 3.5 Hz, 1H),

5.17 (d, J = 4.0 Hz, 1H), 3.8-3.9 (m, 4H), 3.6 – 3.8 (m, 5H), 3.5 - 3.6 (m, 5H), 3.4 - 3.5

(m, 2H), 3.18 (t, J = 9.5 Hz, 1H), 2.5 - 2.6 (m, 1H), 2.26 (t, J = 7.5 Hz, 2H), 1.9 – 2.0 (m,

1H), 1.6 - 1.7 (m, 2H), 1.3 - 1.4 (m, 16H), 0.92 (t, J = 7.0 Hz, 3H). 13C NMR (125 MHz,

CD3OD) δ 175.81, 99.98, 97.53, 84.11, 80.94, 73.69, 72.91, 72.25, 72.05, 71.71, 71.15,

68.78, 66.18, 60.44, 55.54, 49.87, (one carbon is underneath the solvent peak) 39.39,

35.71, 31.67, 29.39, 29.35, 29.28, 29.15, 29.07 29.02, 28.26, 25.68, 22.33, 13.05.

+ + ESI/APCI Calcd for C30H59N4O12 [MH] : 667.4124; Measured m/e: 667.4108.

6′-N-Tetradecanoylkanamycin A (2e). This compound was synthesized as white solid by the reaction of Kanamycin A sulfate with NHS-tetradecyl ester, synthesized as

38 1 before , following the general procedure. H NMR (500 MHz, CD3OD) δ 5.32 (d, J =

4.0 Hz, 1H), 5.15 (d, J = 4.0 Hz, 1H), 3.8-4.0 (m, 5H), 3.6 – 3.7 (m, 4H), 3.4 - 3.6 (m,

7H), 3.18 (t, J = 9.5 Hz, 1H), 2.4 - 2.5 (m, 1H), 2.25 (t, J = 7.5 Hz, 2H), 1.8 – 1.9 (m,

1H), 1.6 - 1.7 (m, 2H), 1.3 - 1.4 (m, 20H), 0.92 (t, J = 7.0 Hz, 3H); 13C NMR (125 MHz,

CD3OD) δ 175.77, 99.91, 98.01, 84.47, 81.93, 73.61, 72.98, 72.43, 72.10, 71.67, 71.21,

68.84, 66.18, 60.37, 55.58, 49.95, (one carbon is underneath the solvent peak), 39.98,

35.68, 31.67, 29.40, 29.38 (3C), 29.36, 29.27, 29.14, 29.07, 29.01, 25.65, 22.32, 13.03.

+ + ESI/APCI Calcd for C32H63N4O12 [MH] : 695.4442; Measured m/e: 695.4418. 89

6′-N-Hexadecanoylkanamycin A (2f). This compound was synthesized as white solid by the reaction of Kanamycin A sulfate with NHS-hexadecyl ester, synthesized as

38 1 before , following the general procedure. H NMR (500 MHz, CD3OD) δ 5.34 (d, J = 3.5

Hz, 1H), 5.17 (d, J = 3.5 Hz, 1H), 3.8 - 4.0 (m, 5H), 3.6 – 3.8 (m, 4H), 3.4 - 3.6 (m, 7H),

3.18 (t, J = 9.5 Hz, 1H), 2.4 - 2.6 (m, 1H), 2.25 (t, J = 7.5 Hz, 2H), 1.8 – 1.9 (m, 1H), 1.6

13 - 1.7 (m, 2H), 1.3 - 1.4 (m, 24H), 0.92 (t, J = 7.0 Hz, 3H); C NMR (125 MHz, CD3OD)

δ 175.78, 99.92, 97.75, 84.28, 81.41, 73.66, 72.97, 72.30, 72.05, 71.68, 71.19, 68.82,

66.17, 60.37, 55.55, 49.88, (one carbon is underneath the solvent peak), 39.97, 35.68,

31.67, 29.40, 29.38 (5C), 29.36, 29.27, 29.14, 29.07, 29.01, 25.65, 22.32, 13.03.

+ + ESI/APCI Calcd for C32H63N4O12 [MH] : 723.4755; Measured m/e: 723.4750.

6′-N-Octadecanoylkanamycin A (2g). This compound was synthesized as white solid by the reaction of Kanamycin A sulfate with NHS-octadecyl ester, synthesized as before38, following the general procedure.1H NMR (500 MHz, CD3OD) δ 5.33 (d, J = 4.0

Hz, 1H), 5.15 (d, J = 3.5 Hz, 1H), 3.8 - 4.0 (m, 5H), 3.6 – 3.8 (m, 4H), 3.4 - 3.6 (m, 7H),

3.18 (t, J = 9.5 Hz, 1H), 2.4 - 2.6 (m, 1H), 2.26 (t, J = 7.0 Hz, 2H), 1.8 – 1.9 (m, 1H), 1.6

- 1.7 (m, 2H), 1.3 - 1.4 (m, 28H), 0.92 (t, J = 7.0 Hz, 3H); 13C NMR (125 MHz, CD3OD)

δ 175.77, 99.93, 98.00, 84.44, 81.90, 73.61, 72.97, 72.44, 72.10, 71.69, 71.20, 68.84,

66.18, 60.38, 55.58, 49.96, (one carbon is underneath the solvent peak), 39.97, 35.68,

31.67, 29.39 (7C), 29.35, 29.35, 29.29, 29.15, 29.07, 29.02, 25.66, 22.33, 13.04.

+ + ESI/APCI Calcd for C36H71N4O12 [MH] : 751.5063; Measured m/e: 751.5057.

6′-N-(2-phenyl-4-quinolinecarbonyl)kanamycin A (2h). To the 2.49 gm (1 eqvt, 10

mmol) of 2-phenylquinoline-4-carboxylic acid dissolved in 50 mL of anhydrous DMF,

1.725 gm (1.5 eqvt, 30 mmol) of N-hydroxysuccinimide and 4.125 gm (2 eqvt. 40 mmol) 90

of N,N'-dicyclohexylcarbodiimide was added and stirred overnight at room temperature.

After filtration of the reaction mixture the solvent from the filtrate was removed by the

air blow. The residue was recrystallized from dichloromethane and ether to obtain pure

2,5-dioxopyrrolidin-1-yl 2-phenylquinoline-4-carboxylate (1h) with 78% yield. 1H NMR

(500 MHz, CDCl3) δ 8.67 (dt, J = 8.5, Hz, 1H), 8.6 (s, 1H), 7.59 (s, 1H), 8.30 (s, J = 8.5

Hz, 1H), 8.24 (d, J = 7.5 Hz, 2H) 7.85 (dt, J = 7.5, 1.5 Hz, 1H), 7.70 (dt, J = 7.5, 1.5 Hz,

13 1H) 7.5 – 7.6 (m, 3H), 3.00 (s, 4H); C NMR (125 MHz, CDCl3) δ 168.92 (2C), 161.57,

156.64, 148.91, 137.97, 131.06, 130.70, 130.37, 130.19, 129.07 (2C), 128.74, 127.63

+ + (2C), 124.90, 123.42, 121.05, 25.78 (2C). ESI/APCI Calcd for C20H15N2O4 [MH] :

347.1032; Measured m/e: 347.1016. Kanamycin A sulfate was reacted with 2,5- dioxopyrrolidin-1-yl 2-phenylquinoline-4-carboxylate as described in the general procedure to give the white solid 6′-N-(2-phenyl-4-quinolinecarbonyl)kanamycin A. 1H

NMR (500 MHz, D2O) δ 7.82 (t, J = 8.0 Hz, 2H), 7.6 – 7.7 (m, 3H), 7.59 (s, 1H), 7.47 (t,

J = 8.0 Hz, 1H), 7.3 – 7.4 (m, 3H), 5.22 (d, J = 3.5 Hz, 1H), 4.29 (d, J = 3.5 Hz, 1H),

4.05 (dd, J = 14.0 Hz, J = 3.5 Hz, 1H), 3.8 – 3.9 (m, 1H), 3.7 – 3.8 (m, 3H), 3.3 - 3.4

(m, 7H), 3.2 – 3.3 (m, 2H), 3.15 (t, J = 11.0 Hz, 1H), 2.99 (t, J = 10.0 Hz, 1H), 2.3 – 2.4

13 (m, 1H), 2.2 – 2.3 (m, 1H) 1.7 – 1.8 (m, 1H); C NMR (125 MHz, D2O) δ 169.49,

157.13, 147.25, 142.75, 137.51, 131.07, 130.22, 129.06 (2C), 127.95, 127.88, 127.56

(2C), 124.77, 122.83, 117.68, 98.66, 95.04, 83.71, 78.31, 72.58, 72.45, 71.51, 71.03,

71.02, 70.97, 67.67, 65.89, 59.74, 54.68, 48.94, 47.36, 40.43, 27.70. ESI/APCI Calcd for

+ + C34H46N5O12 [MH] : 716.3143; Measured m/e: 716.3107.

6′-N-(1-pyrenebutanoyl)kanamycin A (2i). This compound was synthesized as white

solid by the reaction of Kanamycin A sulfate with 2,5-dioxopyrrolidin-1-yl 4-(pyren-1- 91

yl)butanoate, synthesized as before39, following the general procedure.1H NMR (500

MHz, D2O) δ 7.98 (d, J = 7.5 Hz, 1H), 7.95 (d, J = 7.5 Hz, 1H), 7.7 – 7.9 (m, 6H), 7.55

(d, J = 7.5 Hz, 1H), 4.91 (d, J = 3.5 Hz, 1H), 4.77 (d, J = 3.5 Hz, 1H), 3.6 - 3.8 (m, 4H),

3.4 – 3.6 (m, 6H), 3.3 - 3.4 (m, 4H), 2.9 – 3.2 (m, 5H), 2.3 – 2.4 (m, 1H), 2.1 – 2.3 (m,

13 2H) 1.9 – 2.0 (m, 2H), 1.6 - 1.8 (m, 2H); C NMR (125 MHz, D2O) δ 176.86, 135.64,

130.83, 130.32, 129.18, 127.96, 127.35, 127.12, 126.95, 126.33, 126.06, 124.79, 124.74,

124.67, 123.89, 123.86, 123.02, 100.31, 96.93, 83.47, 78.68, 72.69, 72.31, 72.14, 70.98,

70.90, 70.43, 67.99, 65.24, 59.67, 54.96, 49.53, 47.85, 39.47, 35.19, 31.31, 27.48, 26.58.

+ + ESI/APCI Calcd for C38H51N4O12 [MH] : 755.3503; Measured m/e: 755.3476.

6′-N-(fluorescein)kanamycin A (2j). This compound was synthesized as white solid by the reaction of Kanamycin A sulfate with 2,5-dioxopyrrolidin-1-yl 2-(6-hydroxy-3-oxo-

3H-xanthen-9-yl)benzoate, synthesized as before40, following the general procedure. 1H

NMR (500 MHz, D2O) δ 7.77 (d, J = 7.5 Hz, 1H), 7.31 (t, J = 7.0 Hz, 1H), 7.08 (t, J =

7.0 Hz, 1H),6.63 (s, 1H), 6.57 (s, 1H), 6.39 (d, J = 7.5 Hz, 1H ), 6.33 (t, J = 8.5 Hz, 2H),

6.24 (d, J = 9.0 Hz, 1H), 6.18 (d, J = 8.5 Hz, 1H), 5.05 (d, J = 3.5 Hz, 1H), (one proton hidden inside solvent peak), 3.3 – 3.9 (m, 15H), 2.7-2.9, (m, 2H), 2.4-2.5 (m, 1H), 1.8-

13 1.9 (m, 1H); C NMR (125 MHz, D2O) δ 171.94, 157.26, 156.86, 153.39, 152.72,

152.54, 133.80, 129.92, 129.55, 129.02, 128.85, 123.78, 122.82, 112.47, 111.86, 111.27,

108.99, 102.71, 102.35, 100.61, 98.22, 83.53, 79.54, 73.17, 72.66, 71.18, 71.08, 70.51,

69.87, 68.18, 66.20, 65.23, 59.60, 55.03, 49.77, 48.47, 41.28, 27.28. ESI/APCI Calcd for

+ + C38H47N4O15 [MH] : 799.3032; Measured m/e: 799.3037.

6’-N-hexadecanoyltobramycin (3f)

0.20 gm (1 eqvt., 0.50 mmol) of tobramycin and 2 equivalent of potassium carbonate was 92

dissolved in 10 mL of water; then 2 eqvt. of NHS-hexadecyl ester, as synthesized

before38, in 10 mL of DMF was added in 4 portions with one hour of interval. After 48 hours of reaction solvent was removed by air blow and the compounds was purified by column chromatography using 100% MeOH to 10% NH4OH in MeOH.

1 H NMR (500 MHz, D2O) δ 5.38 (d, J = 4.0 Hz, 1H), 5.04 (d, J = 3.5 Hz, 1H), 3.8-3.9

(m, 3H), 3.6 – 3.8 (m, 7H), 3.4 - 3.6 (m, 6H), 3.24 (t, J = 9.0 Hz, 1H), 2.4 - 2.5 (m, 1H),

2.20 (t, J = 7.0 Hz, 2H), 1.8 - 1.9 (m, 1H), 1.4 - 1.6 (m, 2H), 1.1 - 1.3 (m, 4H), 0.79 (t, J =

13 7.0 Hz, 3H); C NMR (125 MHz, D2O) δ 178.04, 100.44, 97.94, 83.55, 79.25, 72.92,

72.73, 72.12, 71.28, 71.10, 70.10, 68.15, 65.34,59.74, 54.99, 49.70, 48.23, 39.10, 35.73,

+ + 30.47, 27.68, 25.12, 21.65, 13.24. ESI/APCI Calcd for C34H68N5O10 [MH] : 706.4966;

Measured m/e: 706.4975.

Procedure for Antibacterial Assay. Antibacterial activity of the compounds was tested

against E. coli (ATCC25922), S. aureus (ATCC25923), S. aureus MRSA (ATCC 33591),

and S. aureus MRSA (ATCC 43300). Bacteria were grown in Lysogeny broth (LB)

medium at 35 oC for 18 hours before using for the assay. The compounds were added in

50 µL with concentration from 512 ug/mL to 0.25 ug/mL with two-fold dilution in 96

well plate. Then 50 µL bacterial growth with scattering OD600 adjusted from 0.08 -0.1

was added in to each well with solution of the compounds and incubated at 35 oC for 18 hours. Then minimum concentration of the compounds which inhibit the growth of the bacterial to the visionary clearance was recorded as the minimum inhibitory concentration of the compound.

Procedure for Antifungal Assay. Antifungal activity of the synthesized compounds was tested against Aspergillus flavus (human and plant pathogen), Fusarium graminearum 93

B4-5A (plant pathogen), Candida albicans 64124 (human pathogen, azole-resistant), C. albicans MYA2876 (human pathogen, azole susceptible), Cryptococcus neoformans H99

(human pathogen), and Rhodotorula pilimanae, using voriconazole as the control following the standard protocol41, 42. RPMI 1640 medium supplemented with 0.165 M

MOPS (pH = 7.0) was used for growing the fungi and during the assay of the compounds. For Fusarium 1x105 cells/mL and for other fungal strain 2x104 cells/mL cell confluence was used during the assay of the compounds. The fungal cells were treated with the compounds from 256 µg/mL to 0.125 µg/mL with two-fold dilution in 96 well plate and incubated for 24 hours to 36 hours. The concentration of the compounds which inhibited the growth of the compounds to clearance was considered as the minimum concentration of the compounds.

Procedure for Membrane Permeabilization Study of 2b and 2g. Candida albicans

MYA2876 cells were grown in potato dextrose broth (PDB) at 28 oC for 48 hours. Cells were spin down at 10,000 rpm for 2 minutes in Fisher Scintific accuSpinTM Micro centrifuge at room temperature and resuspended in the water. Controls (cells only and cells treated with sytox green) along with cells treated with 2b, 2g and sytox green in water were added to different wells of 96 wells plate (Costar 3925) maintaining final volume 200 µL and cell confluence of 1x107 cells/mL. The final concentration of 2b and

2g were 8 µg/mL and the final concentration of sytox was 0.125 0.125 µM. Fluorescent intensity was measured in every 2 minutes for 2 hours with excitation wavelength 485 nm and emission wavelength 528 nm in Cytation 5 imaging reader. Experiment was performed in triplicate. In this study the fluorescence intensity in 60 minutes was similar to the fluorescence in 2 hours, especially the active compound 2g, so we did the imaging 94

of the cells treated with the compounds for 60 minutes in the bright field and green

channels.

Procedure for Kinetic Membrane Permeabilization Study of 2i and 2j. The excitation and emission wavelength of 2j was overlapping with the sytox green dye used in the membrane permeabilization study of 2a and 2g. Therefore, we used propidium iodide

(PI), red fluorescence dye in this study. Propidium iodide function in the same way as sytox green, no or less fluorescence by itself (Figure 4) and the increase in fluorescence upon binding to the nucleic acids (reported in the manuscript). Candida albicans 64124 grown in PDB medium was washed with water and treated with 2i and 2j in water maintaining final concentration of compounds to 1xMIC (16 µg/mL for 2i and 64 µg/mL for 2j) and propidium iodide to 2 µg/mL. Fluorescence intensity was measured in every 2 minutes for 2 hours in Cytation 5 imaging reader with excitation wavelength 538 nm and emission wavelength 617 nm. From this study we see the increase in fluorescence up to 2 hours, especially for 2j, so we did the imaging of the cells incubated for 2 hours in the bright field, blue and red channels.

Procedure for the Fluorescence Imaging of Fungi. Fusarium graminerum spores

(1x105) were grown in PDB medium at 28 oC for 18 hours with gentle shaking. 1 mL of

fungi in the growth was washed twice with water by spinning down at 13000 rpm for 5

minutes in Fisher Scintific accuSpinTM Micro centrifuge at room temperature and

resuspended in the 500 µL of water. Then the 2i and 2j was added to the fungi (16 µg/mL

for 2i and 32 ug/mL for 2j) and incubated at 37 oC for 5 minutes and 60 minutes. After incubation, the cells were washed two times and resuspended in water. Images were taken in blue (for 2i) and green (for 2j) channels in Cyatation 5 imaging reader using 40X 95

objective. Similarly, C. albicans 64124 and C. albicans MYA were grown in PDB medium for 48 hours at 28 oC with gentle shaking. 1 mL of growth was washed with water twice by spinning down at 10000 rpm for 2 minutes in Fisher Scintific accuSpinTM

Micro centrifuge at room temperature and resuspended in the same volume of water.

Then the cells were treated with the 16 µg/mL of 2i and 32 ug/mL of 2j in water

maintaining the cells confluence to 1x107 in 1 mL and incubated at 37 oC for 5 minutes

and 60 minutes. After incubation cells were washed with water two times and

resuspended in the same volume of water. The images of fungi treated with 2i was taken

in Cyation 5 imaging reader in blue channel using 40X objective and the images of the 2j

was taken in Olympus IX71 microscope in green channel using 100X oil immersion

objective.

Procedure for the Assay of Reactive Oxygen Species (ROS) Generation. Candida

albicans MYA grown in potato dextrose broth (PDB) medium was washed with water

and then treated with 1xMIC of 2f, 2g, 2i, K20, 4 (Figure 3) (8 µg/mL for 2f and 2g, 16

µg/mL for 2i and K20, and 4 µg/mL for compound 4), 16 µg/mL of 2b and only media

(control) in RPMI1640 and incubated for 3 hours at 37 oC. The compound 4, known to

35, 36 produce reactive oxygen species, was used as the positive control. After incubation with compounds, cells were washed with water twice and then 25 µM of DCF-DA dye dissolved in RPMI medium was added and incubated for another 30 minutes at 37 oC.

Then the cells were washed with water twice and the fluorescence intensity of the cells measured in Cytation 5 imaging reader with excitation wavelength 485 nm and emission wavelength 525 nm. 96

3.6 References

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11. Chang, C.-W. T.; Fosso, M. Y.; Kawasaki, Y.; Shrestha, S. K.; Bensaci, M. F.; Wang,

J.; Evans, C. K.; Takemoto, J. Y. (2010) Antibacterial to antifungal conversion of

neamine aminoglycosides through alkyl modification. Strategy for reviving old drugs

into agrofungicides. J. Antibiot., 63, 667. DOI: 10.1038/ja.2010.110. 98

12. Fosso, M. Y.; Shrestha, S. K.; Green, K. D.; Garneau-Tsodikova, S. (2015) Synthesis

and bioactivities of kanamycin B derived cationic amphiphiles. J. Med. Chem., 58,

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Membrane lipid-modulated mechanism of action and non-cytotoxicity of novel

fungicide aminoglycoside FG08. PloS one, 8, e73843 DOI

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Increased degree of unsaturation in the lipid of antifungal cationic amphiphiles

facilitates selective fungal cell disruption. ACS Infect. Dis., 4(5), 825-836 DOI

10.1021/acsinfecdis.7b00272.

15. Subedi, Y. P.; AlFindee, M. N.; Takemoto, J. Y.; Chang, C.-W. T. (2018)

Antifungal amphiphilic kanamycins: new life for an old drug. MedChemComm, 9,

909-919 DOI 10.1039/c8md00155c.

16. Chang, C.-W. T.; Takemoto, J. Y. (2014) Antifungal amphiphilic aminoglycosides.

MedChemComm, 5, 1048-105 DOI 10.1039/C4MD00078A.

17. Chang, C.-W. T.; Takemoto, J. Y. (2014) US Patent 8,865,665.

18. Shrestha, S. K.; Chang, C.-W. T.; Meissner, N.; Oblad, J.; Shrestha, J.P.; Sorensen,

K. N.; Grilley, M. M.; Takemoto, J. Y. (2014) Antifungal amphiphilic

aminoglycoside K20: bioactivities and mechanism of action Front. Microbiol., 5, 671

DOI 10.3389/fmicb.2014.00671.

19. Takemoto, J. Y.; Wegulo, S.; Yuen, G. Y.; Stevens, J. L.; Jochum, C. C.; Chang, C.-

W. T.; Kawasaki, Y.; Miller, G. W. (2018) Suppression of wheat Fusarium head 99

blight by novel amphiphilic aminoglycoside fungicide K20. Fungal Biol., 122, 465-

470 DOI 10.1016/j.funbio.2017.12.001.

20. The estimated cost for applying K20 (calculated with chemicals needed for the

synthesis alone) is $60/acre while the costs of agrofungicides in the market, such as

Caramba, Prosaro SC and Headline, are $18.7/acre, $17.9/acre, and $16.0/acre,

respectively. The cost of K20 is calculated based on the cost of chemicals for the

synthesis and amount used for field spraying as reported in reference 13. The cost for

commercial agrofungicides is calculated from the survey of market price reported

from the Institute of Agriculture and Natural Resources , University of Nebraska–

Lincoln (UNL) (https://cropwatch.unl.edu/2017-CW-News/2017-documents/disease-

management/UNL-EC130-Fungicide-Prices-2017.pdf) and the amounts employed for

the field spraying as reported in reference 13.

21. Chandrika, N. T.; Green, K. D.; Houghton, J. L.; Garneau-Tsodikova S. (2015)

Synthesis and Biological Activity of Mono- and Di-N-acylated Aminoglycosides.

ACS Med. Chem. Lett., 6, 1134−1139 DOI: 10.1021/acsmedchemlett.5b00255.

22. Vidotto, V.; Pugliese, A.; Gioannini, P. (1987) Growth of Candida albicans in a

minimal synthetic medium without biotin Mycopathologia., 100, 7-15 DOI

org/10.1007/BF00769562.

23. Pfaller, M. A.; Diekema, D. J.; Colombo, A. L.; Kibbler, C.; Ng, K. P.; Gibbs, D. L.;

Newell, V. A.; and the Global Antifungal Surveillance Group (2006) Candida rugosa,

an Emerging Fungal Pathogen with Resistance to Azoles: Geographic and Temporal

Trends from the ARTEMIS DISK Antifungal Surveillance Program. J. Clin,

Microbiol., 44, 3578-3582 DOI 10.1128/JCM.00863-06. 100

24. Trofa, D.; Gácser, A.; Nosanchuk, J. D. (2008) Candida parapsilosis, an emerging

fungal pathogen. Clin. Microbiol. Rev., 21, 606–625 DOI 10.1128/CMR.00013-08.

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M. J. S. (2013) Candida species: current epidemiology, pathogenicity, biofilm

formation, natural antifungal products and new therapeutic options. J. Med.

Microbiol., 62, 10–24 DOI 10.1099/jmm.0.045054-0.

26. Please refer to appendix B for details.

27. AlFindee, M.; Subedi, Y. P.; Fiori, M.; Krishnan, S.; Kjellgren, A.; Altenberg, G. A.;

Chang, C.-W. T. (2018) Inhibition of Connexin Hemichannels by New Amphiphilic

Amino-glycosides without Antibiotic Activity. ACS Med. Chem. Lett., 9, 7, 697-701

DOI 10.1021/acsmedchemlett.8b00158.

28. Cuccarese, M. F.; Singh, A.; Amiji, M.; O’Doherty, G. A. (2013) A novel use of

gentamicin in the ROS-mediated sensitization of NCI-H460 lung cancer cells to

various anticancer agents. ACS Chem. Biol., 8 (12), 2771-2777DOI

org/10.1021/cb4007024.

29. The information of fluorescence property of 2i and 2j, and other related results can be

found in supporting information.

30. Jaber, Q. Z.; Benhamou, R. I.; Herzog, I.M.; Baruch, B. B.; Fridman, M. (2018)

Cationic amphiphiles induce macromolecule denaturation and organelle

decomposition in pathogenic yeast. Angew. Chem. Int. Ed., 10;57(50):16391-16395

DOI 10.1002/anie.201809410.

31. Please refer to appendix B for detailed images. 101

32. Kohanski, M. A.; Dwyer, D. J.; Hayete, B.; Lawrence, C. A.; Collins, J. J. (2007) A

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Biomed. Opt., 24 (5), 051403 DOI org/10.1117/1.JBO.24.5.051403.

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(2018) Alanine enhances aminoglycosides-induced ROS production as revealed by

proteomic analysis. Front. Microbiol., 9, 29 DOI org/10.3389/fmicb.2018.00029.

35. Shrestha, J. P.; Baker, C.; Kawasaki, Y.; Subedi, Y. P.; Nziko, V. P.; Takemoto, J. Y.;

Chang, C.-W. T. (2017) Synthesis and bioactivity investigation of quinone-based

dimeric cationic triazolium amphiphiles selective against resistant fungal and

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10.1016/j.ejmech.2016.12.008.

36. Shrestha, J. P.; Subedi, Y. P.; Chen, L. L.; Chang, C.-W. T. (2015) MedChemComm,

6, 2012-2022 DOI 10.1039/C5MD00314H.

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CLSI, Wayne, PA, USA.

103

CHAPTER 4

AMPHIPHILIC AMINOGLYCOSIDES AS SELECTIVE INHIBITORS OF CX43

CONNEXIN HEMICHANNEL

4.1 Abstract

Gap junction channels formed by the association of connexin hemichannels (HCs) play a crucial role in intercellular communications. Abnormal opening of the connexin hemichannels is associated with genetic and acquired disorders. Brain, liver, kidney, cardiac muscle, myometrium, and capillary endothelial cells are some of the expression site of Cx43. Uncontrolled opening of the Cx43 HCs mainly affects the heart, including other expression sites of the connexin. Here we have synthesized and tested new aminoglycosides derivatives for their connexin inhibitory activity towards Cx26 and

Cx43. The lead compounds are selective towards Cx43 HCs than Cx26, in contrast to commercial aminoglycosides. These leads have no cytotoxicity up to 100 µM and thus may be used to treat diseases caused by leaky Cx43 HCs. Excellent selectivity and capability to be synthesized on a large scale without a need for column purifications makes 12i the most affordable lead compound for the inhibition of cx43. 104

4.2 Introduction

Gap junction channels (GJCs) formed by the head-to-head association of two

connexin hemichannels (HCs), one from each cell, allow the direct passage of small

molecules, coupling adjacent cells both metabolically and electrically.1-4 The GJCs

accumulate into junctional plaques which allow the intercellular exchange of metabolic

molecules, ions, second messengers, and miRNAs with molecular weight less than 1.5 kDa.5-9 Besides participation in intercellular communication by GJCs formation, HCs also facilitate the communication between extracellular and intracellular milieu in

diseases condition. This kind of HCs opening allows Na+ and Ca2+ ions to enter the cells

and K+ ion, glutamate, and ATP leave the cells. Ischemic conditions,10-11 lower

intracellular redox potential,12 changes in phosphorylation,13-14, etc. can cause the

undesired opening of HCs. Uncontrolled opening of HCs contributes to cell damage, including ischemic damage of the heart (infarct), brain (stroke) and kidneys, some forms

of deafness, and neurodegenerative diseases.15-21

Connexins are expressed at different locations in the cells, and a cell can have

more than one type of connexin.22 Altogether twenty one connexin genes in humans

express 21 types of connexin, among that Cx43 is the most abundant as compared to

Cx26, Cx46, and others.22-23 Cx43 is expressed in variety of organs, such as cardiac

muscle, brain, liver, kidney, myometrium, and capillary endothelial cells.15 Heart disease

is the most common cause of death in the USA, most of these caused by cardiac

ischemia and arrhythmias. Connexins are abundantly expressed in the excitation-

conduction system and the contractile myocardium, and GJCs formed from Cx43

mediate the cell-cell conduction of electrical impulse generated by the sinoatrial 105

node, for the coordinated contraction of the heart.24-27 Cx43 also plays crucial roles in ventricular arrhythmias, including the most lethal one, ventricular fibrillation.24-25, 28-29

For example, embryonic cardiomyocytes transplantation into myocardial infarcts of mice

protected against ventricular tachycardia by the expression of Cx43.30-31 Further, the activation of Cx43 HCs in mammalian cardiomyocytes, astrocytes, and kidney proximal tubule cells under conditions that mimic ischemia contributes to the cell damage.28, 32-35

Connexin inhibitor selective towards Cx43 hemichannel could be a valuable asset

in the treatment of heart disease. Mefloquine, quinine, 2-aminoethoxydiphenyl borate,

glycyrrhetinic acid, flufenamic acid, carbenoxolone, and octanol are some of the previously

reported HCs inhibitors.36-40 Unfortunately, these compounds are either toxic or not selective to HCs. Rationally designed connexin targeted peptides are more promising inhibitors, but they primarily target GJCs, and their clinical use is still not apparent.

A new paradigm in therapeutic development is to “re-purpose” the clinically used

drugs for different applications. Hence, antibacterial AGs have been investigated for their

shown inhibitory activity to connexin HCs (Table 4-1).41-45 These AGs were more

effective in inhibition of Cx26 compared to Cx43 and one other tested connexin HCs.

Our team has previously reported 4″,6″ diarylmethyl kanamycin derivatives. These

amphiphilic kanamycins (AKs) display excellent Cx26 HCs inhibitory activity while

having no antibacterial activity and low toxicity towards human cells (Table 2).46 The

lead compound, 3 was about four-fold more active than the parent kanamycin. More

importantly, several members show selectivity toward Cx43. However, there is no

obvious trend in the structure activity relationship (SAR) that explains the variation of 106 selectivity. Specifically, compounds 1, 2, and 6 are selective towards Cx26 whereas compounds 3, 4, and 5 are more selective towards Cx43. In addition, the necessity of using hazardous sodium azide for the synthesis of an azidoprecursor, azodokanamycin and cumbersome column purification steps limit the large scale synthesis, and increase the difficulty of further investigation of lead compounds. We have recently reported the facile and low-cost synthesis of 6″ and 6′ modified AKs incorporating diverse structural variations. Thus, we decided to explore the HC selectivity of these AKs bearing aryl substituents.

Table 4-1. IC50 values of commercial aminoglycosides towards Cx26 and Cx43

a a a Compound IC50 Cx26 IC50 Cx43 IC50 Cx46

Kanamycin A 11.5 ± 1.8 47.7 ± 1.5 112 ± 5

Kanamycin B 3.6 ± 0.9 24.2 ± 1.9 175 ± 9

Neomycin 7.4 ± 1.3 44.5 ± 8.5 16 ± 2

Paromomycin 41.5 ± 8.6 201.2 ± 17.3 >200

Geneticin (G418) 0.44 ± 0.06 3.0 ± 0.8 1.2 ± 0.2 aUnit = µM 107

Table 4-2. IC50 values of 4″-6″ benzyl substituted kanamycin towards Cx26 and Cx43

NH3 - HO O R = R = HO [Cl ]4 NH3 OH 1 OCH3 O NH HO 3 O 5 O R 2 CH O 3 HO O R H3N 3 Cl 6

4 F

a a b Compound IC50 Cx26 IC50 Cx43 Selectivity

1 13.0 ± 0.7 4.1 ± 1.5 3.17

2 4.3 ± 0.4 3.1 ± 0.9 1.39

3 2.5 ± 0.6 20.3 0.12

4 8.1 ± 0.5 26.6 ± 7.8 0.30

5 6.6 ± 0.5 15 0.44

6 6.2 ± 0.7 5.5 ± 2.0 1.23

a b Unit = µM, selectivity = IC50 Cx26/ IC50 Cx43

108

4.3 Results and Discussion

4.3.1 Synthesis and Design of 6″-Aryl Modified Amphiphilic Kanamycins

We have previously reported the synthesis of 6″-modified AKs via a concise route with the tosylate precursor (compound 9) that could be synthesized in large scale (90 g) as the key intermediate.47 As the aryl entities are the main structural feature of the previously reported 4″,6″ disubstituted kanamycin derivatives, an attempt to incorporate

aryl groups at the 6″ position was implemented. Attempts of nucleophilic substitution

using aryl alcohols and amines were examined and only aryl alcohols (substituted

phenols) furnished the desired products (Scheme 1). Using 9 as the starting material,

compounds 11a-g were synthesized via a nucleophilic substitution followed by the

deprotection of Boc groups. These AKs have structural variations on the electronic effect

of the substituents attached to the phenyl group as well as the steric effect, as in the case

of 11f and 11g.

109

Scheme 4-1. Synthesis of 6″ aryl modified kanamycin A derivatives

NHBoc NHBoc NH2 O HO O O HO HO HO HO NHBoc HO Boc O NHBoc NH2 2 OH Ts-Cl OH OH O NHBoc O NHBoc MeOH, H2O HO O NH2 HO py HO O O O OH O O Ts OH HO O HO HO O OH OH OH BocHN BocHN H N 2 9 54% 8 90% Kanamycin A, (7)

NH3 HO O - HO NH [Cl ]4 1. TFA, DCM OH 3 O NH 2. Ion exchange HO 3 O O O Ts HO OH H3N 10 95%

1. NH HO R 3 O - HO [Cl ]4 HO NH K2CO3, DMF OH 3 o O 60 C, 24 hrs NH3 Compound 9 HO O 2. TFA, DCM O HO O 3. CG50 purification and OH H3N R Ion exchange

R = R = 11a OMe 68% 71% 11f 11b Me 75% 11c H 65% N 11d Cl 63% 11g 41% 58% N 11e NO2 H

4.3.2 Synthesis and design of 6′-modified amphiphilic kanamycins

The synthesis of 6ʹ-modified AKs can be achieved, as reported, in one step from kanamycin with excellent overall yield.48 Following this method, compounds 12a,b which contain an aryl group, have been prepared (Scheme 4-2). 110

Scheme 4-2. Synthesis 6ʹ aryl modified kanamycin derivatives

- O R O NH NH3 HSO4 N O O - HO O 1. HO R HO [Cl ]3 HO NH O NH3 2 7 a - I OH OH O O NH NH3 HO 2 K2CO3 HO O O H2O, DMF OH OH O 2. Acidified with HOAc, HO O HO OH OH Ion exchange H N H2N 3 Kanamycin A sulfate

R = OH OMe 12a, 65% 12b, 75%

In addition to the newly synthesized AKs, we have also selected several AKs that

have been prepared previously to cover more structural features (Figure 4-1).47, 48

NH3 O - HO [Cl ]4 HO NH R = OH 3 O NH O O OH HO 3 O O HN O R HO OH H3N 11h O R' NH - HO O R' = [Cl ]3 HO NH HO O O R' = OH 3 O NH 12f C H HO 3 6 13 O N 12g C7H15 12h C9H19 OH 12c O 12i C11H23 HO OH H3N 12e 12d

Figure 4-1. Structure of previously reported amphiphilic kanamycin

111

4.3.3 Inhibition of amphiphilic kanamycins against connexin

Both 6′ and 6″ modified AKs are tested for their inhibitory activity against Cx26 and Cx43. For the initial screening, two concentrations (15 and 50 µM) are employed using the established E. coli model, as described previously.44-46 The percent inhibition for 6″-modified AKs is shown in Table 4-3, and the percent inhibition for 6′-modified

AKs is shown in Table 4-4. Hydrophobicity of the compounds was calculated in the form of partition coefficient (cLogD) with the goal to explore SAR information.

Nevertheless, based on cLogD, there was no apparent correlation between the IC50 and cLogD, and further investigation will be discussed later.

112

Table 4-3. Percentage of inhibition of Cx26- and Cx43- dependent growth complementation of 6″ modified kanamycin.

Compound % Inhibition Cx26 % Inhibition of Cx43 cLogD

(pH = 7.0) 15 µM 50 µM 15 µM 50 µM

10 26.8 ± 1.7 49.2 ± 5.5 38.1 ± 2.8 51.0 ± 8.8 -12.02

11a 15.9 ± 4.2 36.5 ± 4.7 20.0 ± 7.4 34.8 ± 7.8 -12.52

11b 19.3 ± 2.2 28.2 ± 4.5 63.8 ± 6.8 76.8 ± 5.8 -11.80

11c 14.6 ± 4.3 69.4 ± 4.2 36.0 ± 3.9 46.7 ± 7.8 -12.27

11d 44.4 ± 7.0 69.5 ± 4.5 50.3 ± 1.0 68.3 ± 4.6 -11.75

11e 35.9 ± 2.9 62.6 ± 9.2 36.5 ± 0.6 49.3 ± 2.2 -12.31

11f 46.3 ± 8.6 67.8 ± 6.0 27.3 ± 4.1 54.3 ± 8.9 -10.58

11g 83.1 ± 2.1 85.5 ± 1.7 43.2 ± 7.3 71.7 ± 3.5 -10.88

11h 37.1 ± 13.5 81.0 ± 1.7 45.3 ± 2.7 55.1 ± 5.8 -10.73

113

Table 4-4. Percentage of inhibition of Cx26- and Cx43- dependent growth complementation of 6ʹ modified kanamycin.

Compound % Inhibition of Cx26 % Inhibition of Cx43 cLOgD

(pH = 7.0) 15 µM 50 µM 15 µM 50 µM

12a 4.7 ± 1.6 32.8 ± 11.1 34.1 ± 6.0 37.1 ± 7.4 -10.27

12b 15.1 ± 4.9 27.9 ± 2.6 42.1 ± 1.5 45.1 ± 1.5 -10.24

12c 8.0 ± 2.0 22.8 ± 2.2 25.5 ± 7.1 51.0 ±2.5 -8.81

12d 22.3 ± 6.9 79.6 ± 3.5 56.8 ± 8.8 83.4 ± 1.1 -8.97

12e 36.9 ± 3.9 79.2 ± 7.5 52.2 ± 8.8 69.6 ± 7.5 -7.75

12f 22.5 ± 1.9 34.3 ±2.6 18.5 ± 0.7 36.0 ± 6.1 -10.90

12g 14.0 ± 2.1 25.2 ±2.3 17.0 ± 5.2 37.2 ± 7.6 -10.11

12h 16.1 ±3.2 33.6 ± 3.8 44.4 ± 5.3 53.5 ± 1.9 -9.31

12i 16.4 ± 2.3 56.2 ± 6.8 53.6 ± 4.6 70.9 ± 2.3 -8.52

114

Among these AKs, compounds with relatively higher inhibition, 11b, 11d, and

11g from the 6″ modified AKs and compounds 12d, 12e, and 12i from the 6′ modified

AKs, were selected for the determination of IC50 against Cx26 and Cx43 HCs (Table 4-

5). The IC50 values were calculated from the fittings of Hill’s equation. For the 6″

modified AKs, both compounds 11b and 11d were most active against Cx43, whereas

11g was most active against Cx26, with IC50 at the low micromolar level. Interestingly,

11b was the least active against Cx26, making 11b seven fold more active toward Cx43.

Additionally, 11g was three fold more active toward Cx26. For the 6ʹ modified AKs, all

of the examined compounds had similar activity against Cx43 with IC50 at the low

micromolar level. Since 12i showed the lowest activity against Cx26, a seven fold

selectivity was also observed for 12i toward Cx43. Different from the 6″ modified AKs,

all of the selected 6ʹ modified AKs in this class were more potent against Cx43. Albeit

with the limited number of compounds evaluated with IC50 toward Cx43 and Cx26, the

AKs with 6″ modification showed a correlation between cLogD and selectivity. Those with higher hydrophobicity tend to be more active and selective toward Cx43. The AKs with 6′ modification, however, do not have an obvious correlation, while 6″ modified

AKs may have distinct SAR correlated with cLogD.

115

Table 4-5. IC50 values of 6″ and 6ʹ modified kanamycin towards Cx26 and Cx43 HCs.

a a b Compound IC50 Cx26 IC50 Cx43 Selectivity

11b 49.4 ± 9.3 7.1 ± 1.4 6.96

11d 17.2 ± 3.2 8.9 ± 1.6 1.93

11g 6.0 ± 1.2 17.7 ± 5.6 0.34

12d 12.7 ± 2.2 8.9 ± 3.7 1.42

12e 18.6 ± 2.1 8.6 ± 3.0 2.16

12i 66.7 ± 6.9 9.7 ± 1.8 6.88

a b Unit = µM, Selectivity = IC50 Cx26/ IC50 Cx43

To provide more details, the AKs were divided into two groups (6′ and of 6″ modified) and their percent inhibition toward Cx26 and Cx43 were plotted against cLogD

(Figure 4-2). One of the distinct observations is that there is no correlation between cLogD and the inhibition against Cx26 for both 6′ and of 6″ modified AKs (Figures 4-2A and 4-2C). However, a more definite relationship can be seen towards Cx43 for both 6′ and of 6″ modified AKs (Figures 4-2B and 4-2D). For the 6″ modified AKs, except for

11f-g (circled in Figure 4-2B), those with p-substituted phenyl groups (11a-e) and toluenesulfonyl (10) showed an increase in inhibition against Cx43 with the increase of cLogD (hydrophobicity). Since 11f-g all contain more than one benzene ring, it is likely that the increase in steric hindrance may cause a decrease in the inhibition. 116

For the 6′ modified AKs with linear alkyl groups (compound codes shown in

black) and aryl moieties (compound codes shown in red), a general tendency of

increasing inhibition with the increase of cLogD was observed against Cx43.

Interestingly, an increase in steric hindrance does not seem to lower the inhibition as 12i and 12d were the most active from the AKs with linear alkyl groups and aryl moieties, respectively. Although no apparent SAR can be deduced from the results against Cx26,

AKs having lower cLogD (≤-9.5) exert reduced inhibition. In short, a correlation between the inhibition against Cx43 and cLogD has been found. AKs bearing 6′ and of 6″ modifications have different inhibition profiles. It is possible to enhance the selectivity of

AKs toward Cx43 by tuning the cLogD.

117

Inhibition against Cx26 Inhibition against Cx43 15 µM_Cx26 50 µM_Cx26 15 µM_Cx43 50 µM_Cx43 (A) (B) 11e 11a 11c 10 11b11d 11g 11h 11f 11a 11e11c 10 11b11d 11g 11h 11f 80 80

60 60

6″ modified 40 40 Percentage Inhibition Percentage Inhibition PercentagePercentage Inhibition Inhibition 20 20

-12.5 -12.0 -11.5 -11.0 -10.5 -12.5 -12.0 -11.5 -11.0 -10.5 cLogD cLogD 15 µM_Cx26 50 µM_Cx26

(D) 12f 12a12b12g 12h 12d12c 12i 12e 80 80

60 60

6ʹ modified 40 40

Percentage Inhibition 20 Percentage Inhibition Percentage Inhibition 20 0 -11.0 -10.5 -10.0 -9.5 -9.0 -8.5 -8.0 -7.5 0 cLogD -11.0 -10.5 -10.0 -9.5 -9.0 -8.5 -8.0 -7.5 cLogD

Figure 4-2. Percentage inhibition of Cx26 and Cx43 HC by 6″ and 6 ʹ modified AKs

118

4.3.4 Cytotoxicity of Lead Amphiphilic Kanamycins

The lead AKs were tested for their cytotoxicity using HeLa cells (Figure 4-3). The results showed that these compounds do not manifest significant cytotoxicity at concentrations up to 100 µM.

11b 11d 11g 12d 12e 12i 125 ) M µ

( 100

25 % Cell viability viability Cell % 0 0.1 µM 1.0 µM 10 µM 100 µM

Figure 4-3. Cytotoxicity of lead AKs

4.4 Conclusion

Two libraries of AKs (6′ and of 6″ modified) have been prepared and investigated for their inhibitory activity against Cx26 and Cx43 connexin HCs. For the 6″ modified

AKs, the results showed that the inhibitory effect towards Cx43 was enhanced with the increased hydrophobicity (cLogD) and decreased steric hindrance. For the 6′modified

AKs, a similar correlation between the inhibitory effect and cLogD was noted. However, an increase in steric hindrance did not impede the inhibition. For the activity against

Cx26, no distinct SAR can be derived. In conclusion, the selectivity toward Cx43 can be optimized via the tuning of hydrophobicity of AKs. In addition, the lead AKs showed 119

minimal cytotoxicity against mammalian cells making these classes of compounds ideal

for further studies.

4.5 Experimental Section

General Procedure for the Synthesis of 11a –g. To 0.20 gm (0.2 mmol, 1 eqvt) of

compound 9 dissolved in anhydrous DMF, 2 equiv (0.4 mmol) of substituted phenol and

o 0.055 gm (0.4 mmol, 2 equiv) of K2CO3 were added and heated at 60 C for 24 h with

vigorous stirring. Solvent was removed under reduced pressure and the residue was

washed with 3 X 20 mL water. The washed reaction mixture was dried and treated with 2

mL of trifluoroacetic acid using 10 mL anhydrous DCM as the solvent. The reaction

mixture was stirred at room temperature for 6 h and the solvent was removed by air blow

inside a hood. 10 mL water was added and stirred for 15 minutes at room temperature.

Reaction mixture in water was filtered through a 0.2 µM syringe filter. The water from the filtrate was removed by air blow and the compound was purified by CG50 cation exchange resin (Amberlite). Pure compounds obtained in acetate form after CG50 purification was converted to the chloride form by using IR410 (Cl- form) resin.

6″-O-(4-methoxyphenyl)kanamycin A (11a). Compound was synthesized following the

1 general procedure as a white solid. H NMR (500 MHz, D2O) δ 6.9 – 7.0 (m, 4H), 5.51

(d, J = 3.5 Hz, 1H), 5.05 (d, J = 3.0 Hz, 1H), 4.1 – 4.2 (m, 3H), 3.9 – 4.0 (m, 2H), 3.8 –

3.9 (m, 2H), 3.6 – 3.8 (m, 7H), 3.3 – 3.5 (m, 5H), 3.1 – 3.2 (m, 1H), 2.3 - 2.5 (m, 1H), 1.7

13 - 1.8 (m, 1H); C NMR (125 MHz, D2O) δ 153.63, 152.32, 116.06 (2C), 115.17 (2C),

100.51, 96.82, 84.39, 79.60, 73.00, 72.15, 71.15, 70.87, 70.72, 68.59, 68.20, 66.91, 65.50,

+ + 55.87, 54.96, 50.07, 48.00, 40.27, 28.88. ESI/APCI Calcd for C25H43N4O12 [MH] :

591.2877; measured m/e: 591.2879. 120

6″-O-(4-methylphenyl)kanamycin A (11b). Compound was synthesized following the

1 general procedure as a light-brown solid. H NMR (500 MHz, D2O) δ 7.08 (d, J = 8.5

Hz, 1H), 6.83 (d, J = 8.5 Hz, 1H), 5.50 (d, J = 3.5 Hz, 1H), 5.05 (d, J = 3.5 Hz, 1H), 3.8

– 4.0 (m, 5H), 3.6 – 3.8 (m, 2H), 3.4 – 3.6 (m, 4H), 3.2 – 3.4 (m, 2H), 3.0 – 3.1 (m, 1H),

13 2.4 - 2.5 (m, 1H), 2.14 (s, 1H) 1.8 - 1.9 (m, 1H); C NMR (125 MHz, D2O) δ 155.73,

131.57, 130.20 (2C), 114.78 (2C), 100.59, 96.24, 83.83, 78.15, 72.65, 72.07, 71.18,

70.79, 70.77, 68.64, 68.12, 66.35, 65.47, 54.94, 49.90, 47.84, 40.36, 27.63, 19.53.

+ + ESI/APCI Calcd for C24H47N4O12 [MH] : 575.2928; measured m/e: 575.2927.

6″-O-phenylkanamycin A (11c). Compounds was synthesized following the general

1 procedure as a white solid. H NMR (500 MHz, D2O) δ 7.2 -7.3 (m, 2H), 6.9 – 7.0 (m,

3H), 5.49 (d, J = 4.0 Hz, 1H), 5.05 (d, J = 4.0 Hz, 1H), 4.1 – 4.2 (m, 3H), 3.7 – 4.0 (m,

5H), 3.6 – 3.7 (m, 2H), 3.4 – 3.6 (m, 4H), 3.2 - 3.4 (m, 2H), 3.0 – 3.1 (m, 1H), 2.4 - 2.5

13 (m, 1H), 1.8 - 1.9 (m, 1H); C NMR (125 MHz, D2O) δ 157.87, 129.92 (2C), 121.80,

114.79, 100.57, 96.33, 83.91, 78.37, 72.70, 72.06, 71.11, 70.80, 70.77, 68.61, 68.13,

+ 66.09, 65.48, 54.93, 49.93, 47.88, 40.35, 27.84. ESI/APCI Calcd for C24H41N4O11

[MH]+: 561.2772; measured m/e: 561.2764.

6″-O-(4-chlorophenyl)kanamycin A (11d). Compounds was synthesized following the

1 general procedure as a light-brown solid. H NMR (500 MHz, D2O) δ 7.29 (d, J = 8.5

Hz, 2H), 6.93 (d, J = 9.0 Hz, 2H), 5.52 (d, J = 3.5 Hz, 1H), 5.07 (d, J = 3.0 Hz, 1H), 4.1

– 4.2 (m, 3H), 3.8 – 4.0 (m, 5H), 3.6 – 3.8 (m, 3H), 3.4 – 3.6 (m, 3H), 3.3 – 3.4 (m, 2H),

3.0 – 3.2 (m, 1H), 2.4 - 2.5 (m, 1H), 2.14 (s, 1H) 1.8 - 1.9 (m, 1H); 13C NMR (125 MHz,

D2O) δ 156.64, 129.50 (2C), 125.96, 116.18 (2C), 100.65, 96.67, 83.99, 78.78, 72.81,

72.09, 71.07, 70.80, 70.67, 68.65, 68.15, 66.35, 65.40, 54.91, 49.97, 47.90, 40.26, 28.03. 121

+ + ESI/APCI Calcd for C24H40N4O11 [MH] : 595.2382; measured m/e: 595.2377.

6″-O-(4-nitrophenyl)kanamycin A (11e). Compounds was synthesized following the

1 general procedure as a light-yellow solid. H NMR (500 MHz, D2O) δ 8.19 (d, J = 9.0

Hz, 2H), 7.07 (d, J = 9.0 Hz, 2H), 5.52 (d, J = 3.0 Hz, 1H), 5.05 (d, J = 3.5 Hz, 1H), 4.3

– 4.4 (b, 2H), 4.2 – 4.3 (m, 1H), 3.6 – 4.0 (m, 9H), 3.3 – 3.6 (m, 5H), 3.1 – 3.2 (m, 1H),

13 2.3 - 2.5 (m, 1H), 1.7 - 1.8 (m, 1H); C NMR (125 MHz, D2O) δ 163.52, 141.52, 126.21

(2C), 114.83 (2C), 100.59, 97.26, 84.39, 79.78, 73.19, 72.11, 70.92, 70.83, 70.72, 68.60,

+ 68.22, 66.59, 65.47, 54.91, 50.16, 48.16, 40.27, 29.01. ESI/APCI Calcd for C24H40N5O13

[MH]+: 606.2623; measured m/e: 606.2634.

6″-O-(4-(1,1′-biphenyl))kanamycin A (11f). Compounds was synthesized following the

1 general procedure as a white solid. H NMR (500 MHz, D2O) δ 7.4 – 7.5 (m, 4H), 7.2 –

7.4 (m, 3H), 6.94 (d, J = 8.5 Hz, 2H), 5.51 (d, J = 4.0 Hz, 1H), 5.06 (d, J = 3.5 Hz, 1H),

4.1 – 4.3 (m, 3H), 3.8 – 4.0 (m, 5H), 3.6 – 3.8 (m, 2H), 3.4 – 3.6 (m, 4H), 3.2 – 3.4 (m,

2H), 3.0 – 3.1 (m, 1H), 2.4 - 2.5 (m, 1H), 2.14 (s, 1H) 1.8 - 1.9 (m, 1H); 13C NMR (125

MHz, D2O) δ 157.57, 139.74, 133.73, 129.09 (2C), 128.11 (2C), 127.19, 126.43 (2C),

115.19 (2C), 100.56, 96.67, 83.92, 78.33, 73.03, 72.04, 71.17, 70.86, 70.80, 68.63, 68.14,

+ 66.30, 65.59, 54.98, 49.90, 48.02, 40.38, 27.96. ESI/APCI Calcd for C30H45N4O11

[MH]+: 637.3085; measured m/e: 637.3085.

6″-O-(4-(1H-benzo[d]imidazol-2-yl)phenyl)kanamycin A (11g). 4-(1H-

benzo[d]imidazol-2-yl)phenol was synthesized by refluxing 1,2-diaminobenzene with 4-

hydroxybenzaldehyde in the presence of sodium metabisulfite. 0.475 gm (0.25 mmol, 1

eqvt) of sodium metabisulfite was added to a solution of 0.27 gm (0.25 mmol, 1 eqvt) 122

1,2-diaminobenzene and 0.30 gm (0.25 mmol, 1 eqvt) of 4-hydroxybenzaldehyde in 10 mL DMF. The reaction mixture was refluxed at 150 oC for 24 h. Solvent was removed under reduced pressure and the residue was washed with 2 X 10 mL water followed by 2

X 5 mL DCM. This is previously reported compound.49

NH2 O Na2S2O5 N + OH OH DMF, 150 oC H N NH2 24 h H 4-(1H-benzo[d]imidazol-2-yl)phenol

The compound was synthesized as a white solid following the general procedure using 4-

1 (1H-benzo[d]imidazol-2-yl)phenol as the substituted phenol. H NMR (500 MHz, D2O) δ

7.83 (d, J = 8.0 Hz, 2H), 7.5 – 7.7 (m, 2H), 7.3 – 7.4 (m, 2H), 7.09 (d, J = 8.5 Hz, 2H),

5.58 (d, J = 4.0 Hz, 1H), 5.11 (d, J = 3.5 Hz, 1H), 4.2 – 4.3 (m, 3H), 3.8 – 4.0 (m, 5H),

3.7 – 3.8 (m, 2H), 3.5 – 3.7 (m, 4H), 3.3 - 3.4 (m, 2H), 3.1 – 3.2 (m, 1H), 2.4 - 2.5 (m,

13 1H), 1.8 - 1.9 (m, 1H); C NMR (125 MHz, D2O) δ 161.66, 149.22, 132.19 (2C), 129.28

(2C), 125.59 (2C), 116.16, 115.64 (2C), 113.58 (2C), 100.79, 97.16, 83.85, 78.46, 73.12,

72.01, 71.00, 70.88, 70.72, 68.72, 68.17, 66.23, 65.43, 54.93, 49.95, 48.05, 40.30, 27.72.

+ + ESI/APCI Calcd for C31H45N6O11 [MH] : 677.3146; measured m/e: 677.3148.

6ʹ-N-(6-hydroxy-2-naphthoyl)kanamycin A (12a). Compound was synthesized similar

48 1 to previously reported procedure as a white solid. H NMR (500 MHz, D2O) δ 8.09 (s,

1H), 7.82 (d, J = 9.0 Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.62 (d, J = 8.5 Hz, 1H), 7.18 (s,

1H), 7.14 (d, J = 8.5 Hz, 1H ), 5.34 (d, J = 3.5 Hz, 1H), 4.86 (d, J = 3.0 Hz, 1H), 3.6 –

4.0 (m, 8H), 3.4 – 3.6 (m, 7H), 3.3 – 3.4 (m, 2H), 2.3 – 2.4 (m, 1H), 1.7 – 1.8 (m, 1H); 123

13 C NMR (125 MHz, D2O) δ 171.29, 155.39, 136.33, 131.15, 128.19, 127.89, 127.32,

126.92, 124.05, 119.04, 109. 19, 99.87, 97.45, 83.55, 79.74, 72.66, 72.47, 72.36, 71.47,

71.14, 70.86, 68.07, 65.49, 59.76, 54.86, 49.57, 48.09, 40.56, 27.99. ESI/APCI Calcd for

+ + C29H43N5O13 [MH] : 655.2860; measured m/e: 655.2825.

6ʹ-N-(6-methoxy-2-naphthoyl)kanamycin A (12b). Compound was synthesized similar

48 1 to previously reported procedure as a white solid. H NMR (500 MHz, D2O) δ 8.04 (s,

1H), 7.76 (d, J = 9.5 Hz, 1H), 7.72 (d, J = 9.0 Hz, 1H), 7.62 (d, J = 8.5 Hz, 1H), 7.20 (d,

J = 2.0 Hz, 1H), 7.12 (dd, J = 9.0 Hz, J = 2.5 Hz, 1H ), 5.37 (d, J = 4.0 Hz, 1H), 4.89

(d, J = 3.5 Hz, 1H), 3.8 – 4.0 (m, 5H), 3.4 – 3.8 (m, 13H), 3.3 – 3.4 (m, 2H), 2.4 – 2.5

13 (m, 1H), 1.7 – 1.8 (m, 1H); C NMR (125 MHz, D2O) δ 171.13, 158.50, 136.17, 130.66,

128.34, 127.67, 127.64, 127.32, 124.04, 119.23, 106. 14, 99.97, 97.38, 83.46, 79.30,

72.71, 72.45, 72.31, 71.53, 71.11, 70.78, 68.07, 65.46, 59.77, 55.43, 54.86, 49.56, 48.08,

+ + 40.49, 27.69. ESI/APCI Calcd for C30H45N4O13 [MH] : 669.2983; measured m/e:

669.2985.

Procedure for Large Scale Synthesis of 6ʹ-N-dodecylkanamycin A (12i). To 5.82 gm

(1 equiv, 10 mmol) of kanamycin A sulfate dissolved in 50 mL of water, 2.76 gm (2 equiv, 20 mmol) of anhydrous potassium carbonate was added and stirred for 15 min.

Then 4.46 gm (2 equiv, 20 mmol) of succinimidyl laurate dissolved in 50 mL of dimethylformamide was added slowly in four portions with a 15 minute interval for each addition. After 48 hrs of stirring at room temperature, the solvent was removed by blowing compressed air. The solid residue obtained was washed with 3x50 mL of water.

The white powder collected as a residue was extracted with 5% acetic acid in water to obtain the product. After ion-exchange using IR410 (Cl- form), 6.36 gm (8.2 mmol, 82%) 124

of the desired product was obtained as a white powder. The proton NMR spectra of the

product match with the previously reported spectra.48

Procedure for Cytotoxicity Evaluation. Lead compounds were tested against HeLa,

cervical cancer, cells for their cytotoxicity by colorimetric assay using MTT dye. Cells

o were grown at 37 C at 5% CO2 with a humidified environment. Dulbecco’s modified

eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/mL

penicillin, and 100 µg/mL of streptomycin was used as a growth medium of the cell.

5000 cells in 200 µL growth medium were added to each well of a 96 well cell culture

plate and incubated for 24 hrs. After removing the medium, different concentrations of

compounds (0.0, 0.1, 1.0, 10, and 100 µM) dissolved in the growth medium were added

and incubated for 24 hrs. 20 µL of MTT dye was added to each well and incubated for another 5 hrs. The medium was removed, and the reduced formazan dye was dissolved in

200 µL of DMSO, and the absorption was measured in Bio-Tek imaging reader at 570

nm and 650 nm.

Procedure for the Calculation of cLogD Value. Log D value was calculated keeping

0.1 molar concentration of Na+, K+, and Cl- ionsin Marvin Sketch (version 18.19) . The

values presented are at pH = 7.0. 125

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CHAPTER-5

SUMMARY AND CONCLUSION

A series of 6ʺ modified amphiphilic kanamycin with stable nitrogen and sulfur connector atom were synthesized and tested for biological activity. The SAR study of these compounds showed that the antifungal activity increased with an increase in carbon chain length, reaching optimum with C-14 and C-16 carbon chain. Compounds with both sulfur and nitrogen as the connector atom had a similar trend of biological activity, showing no effect of connector atom. The new leads from these compounds had better activity towards plant and human fungal pathogens, including azole resistant strains, than previously reported lead, K20. The cost of the production of the lead from 6ʺ modified amphiphilic kanamycin series is high, albeit with a stable connector atom, wide-spectrum antifungal activity, and scalability of the synthesis. To resolve this issue, a new series of compounds were synthesized by selective modification at the 6ʹ-NH2 group of the kanamycin A. The SAR of the new compounds was similar to the 6ʺ modified compounds, with C16 and C18 being the optimal chain length. The leads from the new library had wide-spectrum antifungal activity including azole resistant Candida auris. In addition, the lead compounds can be synthesized in a large scale without need of column purification. The mode of action of antifungal compounds is by the fungal membrane permeabilization and generation of reactive oxygen species. Time dependent membrane permeabilization studies showed that these amphiphilic compounds can permeabilize the membrane in less than 5 minutes. Unlike FG08 and K20, the new most active compounds from 6’ and 6” modification also had good antibacterial activity. Although

MIC values towards fungi were much lower than the IC50 values against human cells, 134

these compounds had shown some toxicity towards human cells when treated at 100

µg/mL.

Fluorescence imaging of the fungi, bacteria, and human cells treated with fluorescence analogs revealed that the rate of amphiphilic kanamycin uptake was unique for different types of cells. By controlling the concentration and the incubation time, these fluorescent kanamycins can selectively label pathogenic fungi. In this way, these compounds have the potential to be used as a fluorogenic probe for the selective detection of pathogenic fungi.

A series of previously reported compounds and newly synthesized kanamycin derivatives, with aryl modification at 6″ position and aryl or alkyl modification at 6ʹ position, were screened for their connexin HC inhibitory activity. Among the 6″ modified compounds, those with bulkier substituent group were effective in the inhibition of both Cx26 and Cx43 HCs, while compounds with para-methyl phenyl group selectively inhibited Cx43 HC. The connexin inhibitory activity of the 6ʹ compounds demonstrated that the compounds with more hydrophobic groups have superior activity.

In this series, compounds with dodecyl and 1-pyrenebutyryl substituent groups were most active. Among these two, the compound with a dodecyl group had about seven-fold more selectivity towards Cx43 HC compared to Cx26. These lead compounds did not have toxicity towards human cells, even at up the 100 µM concentrations. The lead compound

6ʹ-N-dodecylkanamycin A, with excellent activity and selectivity towards Cx43, can be synthesized on a large scale by one step modification of kanamycin A.

The leads from both antifungal and connexin inhibitory activity were tested in- vitro. Pharmacokinetic testing of these compounds in animal models can give further 135

insights into the future utility of these compounds. Specifically, testing these compounds in a mouse model could be the future direction of the project. Also, the antifungal AKs with longer carbon chains have slight toxicity and have poor solubility in water at higher concentration, so, work could be directed toward reducing the toxicity of the AKs to human cells and increasing the solubility in water. For this, two shorter alkyl groups could be introduced into the kanamycin core instead of a longer alkyl group.

136

APPENDICES

137

APPENDIX A

CHAPTER 2 SUPPLEMENTARY INFORMATIONS 138

A-1. Excitation and emission spectra of compound 7

1000 Excitation Emission 800 600 RFU 400 200 0 375 450 525 600 675 Wavelenth (nm)

Figure A-1. Excitation and emission fluorescent spectra of compound 7 in water. 139

11 10 9 8 7 6 5 4 3 2 1 0 ppm

.00 2.00 0.92 3.45 0.89 0.83 1.01 0.87 0.52 1.06 1.23

38.48 12.82 Figure A-2. 1H NMR of 6''-O-Toluenesulfonyl-1,3,6',3''-tetra-N-(tert-butoxycarbonyl)kanamycin A (2) in CD3OD 140

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

Figure A-3. 13C NMR of 6''-O-Toluenesulfonyl-1,3,6',3''-tetra-N-(tert-butoxycarbonyl)kanamycin A (2) in CD3OD 141

11 10 9 8 7 6 5 4 3 2 1 0 ppm

2.13 2.15 0.99 2.10 1.01 1.19 1.26 1.12 1.16 2.25 4.32 1.10 1.09 1.13 1.10 1.08 3.25 1.08 1.08 1 Figure A-4. H NMR of 6''-O-Toluenesulfonylkanamycin A (3) in D2O 142

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

Figure A-5. 13C NMR of 6''-O-Toluenesulfonylkanamycin A (3) in D2O 143

11 10 9 8 7 6 5 4 3 2 1 0 ppm

4.25 8.43 1.96 2.27 2.03 6.57 1.00 1.00 1.00 1.98 1.16 1.43 3.25 Figure A-6. 1H NMR of 6″-(1-Hexylamino)-6″-deoxykanamycin A (4a) in D2O 144

220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

Figure A-7. 13C NMR of 6"-(1-Hexylamino)-6"-deoxykanamycin A (4a) in D2O 145

11 10 9 8 7 6 5 4 3 2 1 ppm

2.30 4.74 7.81 2.26 1.09 3.48 2.16 1.19 2.23 1.37 1.37 1.00 1.14

11.73 Figure A-8. 1H NMR of 6”-(1-Octylamino)-6”-deoxykanamycin A (4b) in D2O 146

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

Figure A-9. 13C NMR of 6”-(1-Octylamino)-6”-deoxykanamycin A (4b) in D2O 147

11 10 9 8 7 6 5 4 3 2 1 0 ppm

1.00 1.05 0.96 1.08 1.15 2.05 4.30 4.38 3.91 2.04 1.91 1.87 3.07

14.19

Figure A-10. 1H NMR of 6″-(1-Decylamino)-6″-deoxykanamycin A (4c) in D2O 148

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

Figure A-11. 13C NMR of 6″-(1-Decylamino)-6″-deoxykanamycin A (4c) in D2O 149

11 10 9 8 7 6 5 4 3 2 1 0 ppm

60 1.12 2.24 2.50 1.00 1.06 2.30 4. 3.16 5.70 1.09 2.58 1.11 3.35

19.71

Figure A-12. 1H NMR of 6″-(1-Dodecylamino)-6″-deoxykanamycin A (4d) in D2O 150

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

Figure A-13. 13C NMR of 6″-(1-Dodecylamino)-6″-deoxykanamycin A (4d) in D2O 151

11 10 9 8 7 6 5 4 3 2 1 0 ppm

2 1.77 3.6 7.53 1.75 1.81 2.28 1.07 1.00 0.87 1.02 1.19 3.41

23.69

Figure A-14. 1H NMR of 6”-(1-Tetradecylamino)-6”-deoxykanamycin A (4e) in D2O 152

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

Figure A-17. 13C NMR of 6”-(1-Tetradecylamino)-6”-deoxykanamycin A (4e) in CD3OD 153

11 10 9 8 7 6 5 4 3 2 1 0 ppm

0.70 0.96 4.43 0.65 0.89 4.95 3.29 7.69 6.75 1.36

27.89

Figure A-16. 1H NMR of 6”-(1-Hexadecylamino)-6”-deoxykanamycin A (4f) in CD3OD 154

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

Figure A-17. 13C NMR of 6”-(1-Hexadecylamino)-6”-deoxykanamycin A (4f) in CD3OD 155

11 10 9 8 7 6 5 4 3 2 1 0 ppm

0.91 1.00 4.99 2.04 6.93 0.94 0.87 0.88 0.99 1.86 5.81 2.84 1.87 1.07

Figure A-18. 1H NMR of 6″-(1-Hexylmercapto)-6″-deoxykanamycin A (5a) in D2O 156

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

Figure A-19. 13C NMR of 6″-(1-Hexylmercapto)-6″-deoxykanamycin A (5a) in D2O 157

11 10 9 8 7 6 5 4 3 2 1 0 ppm

Figure A-20. 1H NMR of 6”-(1-Octylmercapto)-6”-deoxykanamycin A (5b) in D2O 158

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

Figure A-21. 13C NMR of 6”-(1-Octylmercapto)-6”-deoxykanamycin A (5b) in D2O 159

11 10 9 8 7 6 5 4 3 2 1 0 ppm

.49 1.01 1.01 3.28 7 1.00 1.13 4.13 3.06 1.96 1.00 1.00 2.02 1.01

14.63 1 Figure A-22. H NMR of 6”-(1-Decylmercapto)-6”-deoxykanamycin A (5c) in D2O 160

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

Figure A-23. 13C NMR of 6”-(1-Decylmercapto)-6”-deoxykanamycin A (5c) in D2O 161

11 10 9 8 7 6 5 4 3 2 1 0 ppm

1.12 1.18 1.06 2.04 1.11 0.94 1.00 4.16 5.45 5.39 1.17 2.05 3.51

19.39

Figure A-24. 1H NMR of 6”-(1-Dodecylmercapto)-6”-deoxykanamycin A (5d) in D2O 162

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

Figure A-25. 13C NMR of 6”-(1-Dodecylmercapto)-6”-deoxykanamycin A (5d) in D2O 163

11 10 9 8 7 6 5 4 3 2 1 ppm

1.00 1.10 1.31 1.98 1.16 1.96 3.31 2.35 6.81 2.97 1.11 1.98 3.51

23.46

Figure A-26. 1H NMR of 6”-(1-Tetradecylmercapto)-6”-deoxykanamycin A (5e) in CD3OD 164

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

Figure A-27. 13C NMR of 6”-(1-Tetradecylmercapto)-6”-deoxykanamycin A (5e) in CD3OD 165

11 10 9 8 7 6 5 4 3 2 1 0 ppm

1.38 1.86 3.14 1.96 2.60 1.22 2.11 4.46 2.14 1.76 1.04 1.00 1.19 3.18

27.08 1 Figure A-28. H NMR of 6”-(1-Hexadecylmercapto)-6”-deoxykanamycin A (5f) in CD3OD 166

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

Figure A-29. 13C NMR of 6”-(1-Hexadecylmercapto)-6”-deoxykanamycin A (5f) in CD3OD 167

11 10 9 8 7 6 5 4 3 2 1 ppm

9 0.91 1.94 0.98 1.00 1.06 1.08 1.68 0.76 1.6 0.92 1.34 7.32 2.63 1.37 0.88 3.19 1.97

Figure A-30. 1H NMR of 6″-(2-(6-hydroxy-3-oxo-3H-xanthen-9-yl))benzamido)-6″-deoxykanamycin A (7) in D2O 168

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm Figure A-31. 13C NMR of 6″-(2-(6-hydroxy-3-oxo-3H-xanthen-9-yl))benzamido)-6″-deoxykanamycin A (7) in D2O 169

ppm

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 ppm

Figure A-32. 1H-1H COSY of 6''-O-Toluenesulfonylkanamycin A (3) in D2O 170

6" 4" 3" 1' 1" 3' 2' 4' 2" 6'e 6'a 5" 5'

ppm

3.2

3.4

3.6

3.8

4.0

4.2

4.4

4.6

4.8

5.0

5.2

5.4

5.6 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 ppm

Figure A-33. 1H-1H COSY of 6''-O-Toluenesulfonylkanamycin A (3) (zoomed version) in D2O 171

APPENDIX B

CHAPTER 3 SUPPLEMENTARY INFORMATIONS 172

B-1. Excitation and Emission Spectra of 2i and 2j.

Excitation and emission fluorescence spectra of the 2i and 2j were measured in

Spectrofluorophotometer (SHIMADZU, RF-5301PC) at 0.1 µg/mL and 0.3 µg/mL final

concentration respectively in water. For excitation spectra of the 2i, emission wavelength

was fixed at 380 nm and the compound was excited at 340 nm for emission spectra.

Similarly, for excitation spectra of 2j, emission wavelength was fixed at 525 nm and for

emission spectra the compound was excited at 485 nm.

100 Excitation Emission 100 Excitation Emission 80 80 60 60 RFU 40 RFU 40 20 20 0 0 325 350 375 400 425 450 475 500 525 550 575 600 Wavelength (nm) Wavelength (nm) A [B]

Figure B-1: Excitation and emission fluorescent spectra of 2i [A] and 2j [B] in water

B-2. Images of Candida albicans Treated with Propidium Iodide

Candida albicans 64124 was treated with 2i and 2j along with PI and for control the fungi was treated with propidium iodide only. 173

Figure B-2: Image of Candida albicans 64124 treated with PI after 2 hours incubation

B-3. Imaging of Mammalian Cells

HeLa cells were grown in DMEM medium as described in cytotoxicity evaluation

study. Then 10,000 cells in 400 µL medium were seeded in Lab-Tek II chambered cover

o glass (155409) and incubated for 24 hours at 37 C in 5% CO2 atmosphere. Cells were

washed with 1xPBS (PH = 7.4) and 32 µg/mL of KI compound in DMEM medium was

added to the cells. After 5 minutes or 60 minute of incubation cells were washed with

PBS and images were taken under bright field and green channels.

Figure B-3: Images of mammalian cells treated with 2j. Hela cells incubated with 2j at 32

µg/mL for 5 min. (A and B) and 60 min. (C and D) 174

B-4. Imaging of Bacteria

Bacterial cells (E. coli (ATCC25922), S. aureus (ATCC25923)) were grown in

LB medium at 35 oC for 18 hours. 1 mL of the bacterial cells were washed with water by

centrifuging at 10,000 rpm for 2 minutes in Fisher Scintific accuSpinTM Micro centrifuge.

Then bacteria were resuspended in the solution of 2j maintaining the final concentration

to 32 µg/mL and 128 µg/mL for each bacteria type. Bacteria were incubated for 5

minutes and 60 minutes at 37 oC, washed with water twice and finally resuspended in

water. The images of the bacteria were taken in Olympus IX71 microscope in green

channel using 100X oil immersion objective. 175

Figure S3-4: Images of bacteria treated with 2j. (A) – (D): E. coli incubated with 2j at

32 µg/mL for 5 min. (A and B) and 60 min. (C and D); (E) – (H): E. coli incubated with

2j at 128 µg/mL for 5 min. (E and F) and 60 min. (G and H); (I) – (L): S. aureus incubated with 2j at 32 µg/mL for 5 min. (I and J) and 60 min. (K and L); (M) – (P): S. aureus incubated with 2j at 128 µg/mL for 5 min. (M and N) and 60 min. (O and P).

176

B-5. Cytotoxicity Assay of the Compounds

Cytotoxicity of the compounds was evaluated against HeLa cells, cervical human cells by colorimetric assay using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) dye. Cells were grown in DMEM medium (Corning) supplemented with 10% fetal bovine serum, 100 U/mL of penicillin and 100 U/mL of streptomycin at 37 oC under 5% CO2 environment. 5000 cells in 200 µL of medium were

added to the 96 well cell culture plate (Corning) and incubated for 24 hours to let the

cells attach to the plate. Medium was removed and different concentration of compounds

(0.1, 1.0, 10, 100) and the medium only, as a control, was added to the cells. After 24

hours of incubation 20 µL of MTT dye (20 mg/mL) was added and incubated for another

5 hours. Then the medium was removed and the reduced form of MTT was dissolved in

200 µL DMSO. The absorption of the dissolved solution was taken in 570 nm using 650

nm as the background absorption. Percentage cell viability of the cells at different

concentration of compounds was expressed relative to the control, which was considered

as the 100 percent cell viability.

177

2d 2e 2f 2g 3f 2i 2j FG08 K20

100

50 % Cell viability Cell % 25

0 0.1 µg/mL 1.0 µg/mL 10 µg/mL 100 µg/mL

Figure B-5: Cell viability of the HeLa cells at different concentration of the compounds

Table B-1. IC50 values of the compounds

Compound IC50 value Compound IC50 value

(µg/mL) (µg/mL)

2d >100 2i >100

2e 67.41±3.91 2j >100

2f 64.61±1.77 K20 >100

2g 62.96±2.35 FG08 >100

3f 51.90±4.31

178

11 10 9 8 7 6 5 4 3 2 1 0 ppm

1.06 2.03 1.02 1.00 0.95 1.03 1.93 1.07 4.32 1 Figure B-6. 500 MHz H -NMR spectrum of 2,5-dioxopyrrolidin-1-yl 2-phenylquinoline-4-carboxylate (1h) in CDCl3

179

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

13 Figure B-7. 125 MHz C -NMR spectrum of 2,5-dioxopyrrolidin-1-yl 2-phenylquinoline-4-carboxylate (1h) in CDCl3

180

11 10 9 8 7 6 5 4 3 2 1 0 ppm

1.01 1.00 3.18 1.15 2.17 1.04 3.31 7.84 6.65 2.22 4.53 1.08 1 Figure B-8. 500 MHz H-NMR spectrum of 6'-N-Hexanoylkanamycin A (2a) in D2O 181

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm 13 Figure B-9. 125 MHz C-NMR spectrum of 6'-N-Hexanoylkanamycin A (2a) in D2O 182

11 10 9 8 7 6 5 4 3 2 1 0 ppm

1.07 7.62 6.44 1.00 0.98 3.14 1.11 2.06 1.01 2.09 8.46 3.09 1 Figure B-10. 125 MHz H-NMR spectrum of 6'-N-Octanoylkanamycin A (2b) in D2O 183

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

13 Figure B-11. 125 MHz C-NMR spectrum of 6'-N-Octanoylkanamycin A (2b) in D2O 184

11 10 9 8 7 6 5 4 3 2 1 0 ppm

1.29 6.26 7.83 1.84 0.93 1.06 3.13 1.26 1.79 0.95 2.72

11.09 1 Figure B-12. 500 MHz H-NMR spectrum of 6'-N-Decanoylkanamycin A (2c) in D2O 185

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm 1 Figure B-13. 125 MHz 3C-NMR spectrum of 6'-N-Decanoylkanamycin A (2c) in D2O 186

11 10 9 8 7 6 5 4 3 2 1 0 ppm

2.24 0.94 4.17 4.92 0.95 2.06 0.98 1.95 3.06 1.00 4.86 0.81

16.55 1 Figure B-14. 500 MHz H-NMR spectrum of 6'-N-Dodecanoylkanamycin A (2d) in CD3OD 187

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

13 Figure B-15. 125 MHz C-NMR spectrum of 6'-N-Dodecanoylkanamycin A (2d) in CD3OD 188

11 10 9 8 7 6 5 4 3 2 1 0 ppm

1.00 5.05 7.17 1.14 1.16 2.11 1.12 2.09 3.07 1.08 4.01

20.30 1 Figure B-16. 500 MHz H-NMR spectrum of 6'-N-Tetradecanoylkanamycin A (2e) in CD3OD 189

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

13 Figure B-17. 125 MHz C-NMR spectrum of 6'-N-Tetradecanoylkanamycin A (2e) in CD3OD 190

11 10 9 8 7 6 5 4 3 2 1 0 ppm

0.90 1.00 4.10 7.29 1.12 1.08 1.83 1.05 2.12 3.16 5.12

24.54 1 Figure B-18. 500 MHz H-NMR spectrum of 6'-N-Hexadecanoylkanamycin A (2f) in CD3OD 191

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

13 Figure B-19. 125 MHz C-NMR spectrum of 6'-N-Hexadecanoylkanamycin A (2f) in CD3OD 192

11 10 9 8 7 6 5 4 3 2 1 0 ppm

7.68 1.10 1.09 4.25 2.15 2.39 5.40 1.16 1.44 1.00 3.90

33.10 1 Figure B-20. 500 MHz H-NMR spectrum of 6'-N-Octadecanoylkanamycin A (2g) in CD3OD 193

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

13 Figure B-21. 125 MHz C-NMR spectrum of 6'-N-Octadecanoylkanamycin A (2g) in CD3OD 194

11 10 9 8 7 6 5 4 3 2 1 0 ppm

2.02 3.03 0.96 1.01 3.05 1.00 0.98 0.97 1.03 3.15 7.30 1.99 1.01 0.96 1.04 0.97 1.00 1 Figure B-22. 500 MHz H-NMR spectrum of 6'-N-(2-phenyl-4-quinolinecarbonyl)kanamycin A (2h) in D2O 195

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

13 Figure B-23. 125 MHz C-NMR spectrum of 6'-N-(2-phenyl-4-quinolinecarbonyl)kanamycin A (2h) in D2O 196

11 10 9 8 7 6 5 4 3 2 1 0 ppm

1.04 1.01 6.12 1.01 0.95 1.06 4.09 5.38 1.36 4.03 3.61 2.27 1.12 2.23 2.07 1.18

1 Figure B-24. 500 MHz H-NMR spectrum of 6'-N-(1-Pyrenebutanoyl)kanamycin A (2i) in D2O 197

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

13 Figure B-25. 125 MHz C-NMR spectrum of 6'-N-(1-Pyrenebutanoyl)kanamycin A (2i) in D2O 198

11 10 9 8 7 6 5 4 3 2 1 0 ppm

1.00 0.99 0.86 0.90 0.93 1.01 1.71 0.92 0.89 0.91 0.94 2.74 1.07

15.28

1 Figure B-26. 500 MHz H-NMR spectrum of 6'-N-(Fluorescein)kanamycin A (2j) in D2O 199

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

13 Figure B-27. 125 MHz C-NMR spectrum of 6'-N-(Fluorescein)kanamycin A (2j) in D2O 200

11 10 9 8 7 6 5 4 3 2 1 0 ppm

7.77 1.56 1.25 2.64 2.08 1.42 1.80 1.00 1.23 7.56 1.23 3.08

25.68 1 Figure B-28. 500 MHz H-NMR spectrum of 6'-N-Hexadecanoyltobramycin (3f) in CD3OD 201

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

13 Figure B-29. 125 MHz C-NMR spectrum of 6'-N-Hexadecanoyltobramycin (3f) in CD3OD 202

11 10 9 8 7 6 5 4 3 2 1 0 ppm

93 0.99 1.00 3.05 1.02 1.18 1. 4.41 1.10 1.10 1.13 3.13 1.02 0.97 0.99 1.81 0.97 2.02 5.24

1 Figure B-30. 500 MHz H-NMR spectrum of 6'-N-Hexanoylkanamycin A (2a) (neutral form) in D2O 203

ppm

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

Figure B-31. 500 MHz 1H-1H COSY spectrum of 6'-N-Hexanoylkanamycin A (2a) in D2O 204 205

APPENDIX C

CHAPTER 4 SUPPLEMENTARY INFORMATIONS 206

C-1. Caluculation of IC50 value.

To determine inhibition of connexin hemichannels by the new compounds we first evaluated the effects at 15 and 50 µM. These concentrations correspond to the IC50 of kanamycin A for the inhibition of Cx26- and Cx43-dependent growth, respectively.

The idea was to select for compounds that are at least as good as previous ones, and then determine the values of IC50 of promising compounds, emphasizing those that show improvements for potency against Cx43. IC50 values were caluculated from the fitting’s of the Hill’s equation to the data.

Figure C-1. Inhibition of Cx26 dependent growth complementation by kanamycin derivatives 207

Figure C-2. Inhibition of Cx43 dependent growth complementation by kanamycin derivatives 208

11 10 9 8 7 6 5 4 3 2 1 ppm

1.18 4.05 1.15 1.00 2.94 2.16 1.88 7.23 5.53 1.10 1.21

1 Figure C- 3. H-NMR of 6″-O-(4-methoxyphenyl)kanamycin A (11a) in D2O 209

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

13 Figure C- 4. C-NMR of 6″-O-(4-methoxyphenyl)kanamycin A (11a) in D2O 210

11 10 9 8 7 6 5 4 3 2 1 ppm

1.95 1.94 1.00 1.18 3.02 5.54 2.33 4.40 2.16 1.00 1.09 2.86 1.01

1 Figure C-5 . H-NMR of 6″-O-(4-methylphenyl)kanamycin A (11b) in D2O 211

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

13 Figure C-6 . C-NMR of 6″-O-(4-methylphenyl)kanamycin A (11b) in D2O 212

11 10 9 8 7 6 5 4 3 2 1 ppm

1.90 2.82 1.01 1.12 3.00 5.73 2.29 4.61 2.40 1.12 1.26 1.41

Figure C-7 . 1H-NMR of 6″-O-phenylkanamycin A (11c) in D2O 213

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

13 Figure C-8. C-NMR of 6″-O-phenylkanamycin A (11c) in D2O 214

11 10 9 8 7 6 5 4 3 2 1 ppm

1.29 1.47 1.89 1.91 0.97 1.00 3.08 5.56 2.43 1.17 3.36 2.10 1.06

1 Figure C-9. H-NMR of 6″-O-(4-chlorophenyl)kanamycin A (11d) in D2O 215

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

13 Figure C-10. C-NMR of 6″-O-(4-chlorophenyl)kanamycin A (11d) in D2O 216

11 10 9 8 7 6 5 4 3 2 1 ppm

5 1.83 1.89 0.98 1.00 1.8 1.00 9.16 5.26 1.09 1.13 1.16

1 Figure C-11. H-NMR of 6″-O-(4-nitrophenyl)kanamycin A (11e) in D2O 217

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

13 Figure C-12. C-NMR of 6″-O-(4-nitrophenyl)kanamycin A (11e) in D2O 218

11 10 9 8 7 6 5 4 3 2 1 0 ppm

3.91 2.13 1.15 1.93 1.00 1.05 3.04 2.45 3.43 2.58 4.24 2.40 0.95 1.20 1.24

1 Figure C-13. H-NMR of 6″-O-(4-(1,1′-biphenyl))kanamycin A (11f) in D2O 219

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

13 Figure C-14. C-NMR of 6″-O-(4-(1,1′-biphenyl))kanamycin A (11f) in D2O 220

11 10 9 8 7 6 5 4 3 2 1 0 ppm

1.77 2.00 2.03 1.88 1.00 1.16 1.02 1.04 2.92 5.11 2.23 4.61 2.14 0.99

Figure C-15. 1H-NMR of 6″-O-(4-(1H-benzo[d]imidazol-2-yl)phenyl)kanamycin A (11g) in D2O 221

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

Figure C-16. 13C-NMR of 6″-O-(4-(1H-benzo[d]imidazol-2-yl)phenyl)kanamycin A (11g) in D2O 222

11 10 9 8 7 6 5 4 3 2 1 0 ppm

0.96 2.13 0.96 0.95 0.80 7.43 0.89 0.98 0.98 1.02 1.13 7.71 1.27 1.05

1 Figure C-17. H-NMR of 6 -N-(6-hydroxy-2-naphthoyl)kanamycin A (12a) in D2O 223

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

13 Figure C-18. C-NMR of 6 -N-(6-hydroxy-2-naphthoyl)kanamycin A (12a) in D2O 224

11 10 9 8 7 6 5 4 3 2 1 ppm

0.92 0.95 0.96 0.90 0.93 0.94 0.98 1.03 1.17 4.35 6.61 3.45 4.35 2.23 1.11 1.20

1 Figure C-19. H-NMR of 6'-N-(6-methoxy-2-naphthoyl)kanamycin A (12b) in D2O 225

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

13 Figure C-20. C-NMR of 6'-N-(6-methoxy-2-naphthoyl)kanamycin A (12b) in D2O 226

APPENDIX D

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Scalable and cost-effective tosylation-mediated synthesis of antifungal and fungal diagnostic 6″-Modified amphiphilic kanamycins Author: Yagya Prasad Subedi,Uddav Pandey,Madher N. Alfindee,Heath Montgomery,Paul Roberts,Jeffrey Wight,Gavin Nichols,Michell Grilley,Jon Y. Takemoto,Cheng-Wei Tom Chang Publication: European Journal of Medicinal Chemistry Publisher: Elsevier Date: 15 November 2019

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Development of fungal selective amphiphilic kanamycin: Cost-effective synthesis and use of fluorescent analogs for mode of action investigation, Yagya Prasad Subedi, Paul Roberts, Michelle Grilley, Jon Y. Takemoto, and Cheng-Wei Tom Chang, ACS Infect. Dis. 2019, 5 (3), 473-483.

Scalable and cost-effective tosylation-mediated synthesis of antifungal and fungal diagnostic 6 ″-Modified amphiphilic kanamycins, Yagya Prasad Subedi, Uddav Pandey, Madher N. Alfindee, Heath Montgomery, Paul Roberts, Jeffrey Wight, Gavin Nichols, Michell Grilley, Jon Y Takemoto, Cheng-Wei Tom Chang, Eur. J. Med. Chem. 2019, 182, 111639. A copy of this permission letter will be included in my dissertation appendix. Please indicate your approval by signing in the provided space, attaching any other form or instruction necessary to confirm permission.

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Development of fungal selective amphiphilic kanamycin: Cost-effective synthesis and use of fluorescent analogs for mode of action investigation, Yagya P. Subedi, Paul Roberts, Michelle Grilley, Jon Y. Takemoto, and Cheng-Wei Tom Chang, ACS Infect. Dis. 2019, 5 (3), 473-483.

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CURRICULUM VITAE

YAGYA P. SUBEDI

0300 old main hill Logan, UT, 84322 Email: [email protected] Phone: (435) 213 0168

EDUCATION 2013 – 2019 Ph.D., Organic Chemistry, Utah State University, Logan, UT. Advisor: Prof. Cheng-Wei Tom Chang 2007 – 2011 M.S., Organic Chemistry, Tribhuvan University, Kathmandu, Nepal, 2011. Advisor: Prof. Susan Joshi

SKILLS

• Multistep synthesis

• Synthesis of carbohydrate and heterocyclic compounds

• Milligram to kilogram scale synthesis

• Drug discovery, SAR study

• Hit to lead optimization

• 1D and 2D NMR, IR, MS

• HPLC, LC-MS, GC

• UV-Vis, Fluorescence imaging/assay

• Cell-based Bioassay: anticancer, antibacterial, and antifungal assay

• Creative, Self-dependent, excellent leader

• Microsoft office, Sci-finder, Chemdraw, Marvin sketch 238

AWARDS AND HONOR

• Outstanding Graduate Student in Chemistry, 2019

• RGS Graduate Student Travel Award, 2019

• Dr. Leslie Paxton Barker Travel Award, 2019

MEMBERSHIP

• American Chemical Society

• American Association for the Advancement of Science

RESEARCH EXPERIENCE

Graduate Research Assistant, Utah State University 2013 - 2019

• Synthesis of a library of cationic anthraquinone analogs selective towards cancers and

resistance gram-positive bacteria and identification of lead from SAR study.

• Cost-effective synthesis of bioactive amphiphilic kanamycin and investigation of

detail mode of action using newly synthesized fluorescent analogs.

• Synthesis of fluorescent aminoglycosides which can selectively stain the fungi.

• Synthesis of new aminoglycosides selective towards Cx43 connexin hemichannel

inhibition which are a potential lead for the treatment of heart disease.

• Synthesis of fluorescent lignin probes and establish a new bioassay protocol to study

the fragmentation of lignin using mutated white rote fungi. The newly obtained

mutant can degrade the lignin more effectively than wild type.

• Working on the synthesis of carbon quantum dots from the natural products for the

detection of fungi.

• Served in the departmental student safety committee. 239

M.S., Organic chemistry, Tribhuvan University 2007- 2011

• Extraction, isolation and purification of the bioactive compounds from the heartwood

of Acacia catechu.

• Bioactivity of the different extracts and the isolated compounds were evaluated.

TEACHING EXPERIENCE

Graduate Teaching Assistant, Utah State University 2013 – 2019

• Prepared lab manual and taught advance synthesis CHEM5530 (Spring 2019)

• Teaching assistant for organic chemistry I and II (2016– 2019)

• Teaching assistant for quantitative analysis CHEM3005 (Fall 2014)

• Teaching assistant for general chemistry I and II (2013 Fall, 2014 Spring)

• Mentored 13 undergraduate and 2 graduate research assistants

Lecturer, Pinnacle College, Kathmandu, Nepal 2011-2013

• Taught general chemistry and organic chemistry to high school and undergraduate

classes

PUBLICATIONS

17. Mangum, C. L.; Munford, M. B.; Sam, A. B.; Young, S. K.; Beales, J. T.; Subedi, Y.

P.; Mangum, C. D.; Allen, T. J.; Liddella, M. S.; Merrell, A. I.; Saavedra, D. I.;

Williams, B. J.; Evans, N.; Beales, J. L.; Christiansen, M. A., The total syntheses of

JBIR-94 and two synthetic analogs and their cytotoxicities against A549 (CCL-185)

human small lung cancer cells. Tetrahedron Lett. 2019. (Just accepted)

16. Pandey, U.; Subedi, Y. P.; Alfindee, M. N.; Shepherd, T.; Chang, C.-W. T. An

Alternative and facile synthetic approach for the precursors of 3- and 6-aminosugar 240

donors and study of one-pot glycosyltrasferation. (Just accepted)

15. Subedi, Y. P.; Pandey, U.; Alfindee, M. N.; Montgomery, H.; Roberts, P.; Wight, J.;

Nichols, G.; Grilley, M.; Takemoto, J. Y.; Chang, C.-W. T. Scalable and cost-

effective tosylation-mediated synthesis of antifungal and fungal diagnostic 6″-

modified amphiphilic kanamycins. Euro. J. Med. Chem. 2019, 182,

doi.org/10.1016/j.ejmech.2019.111639.

14. AlFindee, M. N.*; Subedi, Y. P.*; Grilley, M.; Takemoto, J. Y.; Chang, C.-W. T.

Antifungal property of 4″,6″-disubstituted amphiphilic kanamycins.

Molecules 2019, 24(10), 1882. *Co first author.

13. Subedi, Y. P.; Chang, C.-W. T., Cationic anthraquinone analogs as selective

antimicrobials. Microbiol. Insights. 2019, 12, 1-4.

12. Subedi, Y. P.; Roberts, P.; Grilley, M.; Takemoto, J. Y.; Chang, C.-W. T.

Development of fungal selective amphiphilic kanamycin: cost effective synthesis and

use of fluorescent analogs for mode of action investigation. ACS Infect. Dis. 2019, 5,

3, 473-783.

11. Kjellgren, A.; Fiori, M.; AlFindee, M.; Subedi, Y. P.; Krishnan, S.; Chang, C.-W. T.;

Altenberg, G. A.; Inhibition of connexin hemichannels by new amino-glycosides

without antibiotic activity. Biophys. J. 2019, 116(3), 250a.

10. Subedi, Y. P.; Alfindee, M. N.; Shrestha, J. P.; Chang, C.-W. T., Tuning the

biological activity of cationic anthraquinone analogues specifically toward

Staphylococcus aureus. Euro. J. Med. Chem. 2018, 157, 683-690.

9. Subedi, Y. P.; Alfindee, M. N.; Shrestha, J. P.; Becker, G.; Grilley, M.; Takemoto, J.

Y.; Chang, C.-W. T., Synthesis and biological activity investigation of azole and 241

quinone hybridized phosphonates. Bioorg. Med. Chem. Lett. 2018, 28 (18), 3034-

3037.

8. AlFindee, M.; Subedi, Y. P.; Fiori, M.; Krishnan, S.; Kjellgren, A.; Altenberg, G. A.;

Chang, C.-W. T., Inhibition of Connexin Hemichannels by New Amphiphilic Amino-

glycosides without Antibiotic Activity. ACS Med. Chem. Lett. 2018.

7. Subedi, Y. P.; AlFindee, M. N.; Takemoto, J. Y.; Chang, C.-W. T., Antifungal

amphiphilic kanamycins: new life for an old drug. MedChemComm 2018, 9 (6), 909-

919.

6. AlFindee, M. N.; Zhang, Q.; Subedi, Y. P.; Shrestha, J. P.; Kawasaki, Y.; Grilley,

M.; Takemoto, J. Y.; Chang, C.-W. T., One-step synthesis of carbohydrate esters as

antibacterial and antifungal agents. Bioorg. Med. Chem. 2018, 26 (3), 765-774.

5. Shrestha, J. P.; Baker, C.; Kawasaki, Y.; Subedi, Y. P.; de Paul, N. N. V.; Takemoto,

J. Y.; Chang, C.-W. T., Synthesis and bioactivity investigation of quinone-based

dimeric cationic triazolium amphiphiles selective against resistant fungal and

bacterial pathogens. Euro. J. Med. Chem. 2017, 126, 696-704.

4. Li, X.; Gui, S.; Bhuiyan, M.; Zeng, W.; Subedi, Y.; Want, R.; Chen, L., Quantifying

uptake and retention of copper ions in silica-encrusted Chlamydomonas reinhardtii.

Biochem. Anal. Biochem. 2015, 4: 228. Doi: 10.4172/2161-1009.1000228.

3. Shrestha, J. P.; Subedi, Y. P.; Chen, L.; Chang, C.-W. T., A mode of action study of

cationic anthraquinone analogs: a new class of highly potent anticancer agents.

MedChemComm 2015, 6 (11), 2012-2022. 242

2. Lamichhane, P.; Joshi, D.; Subedi, Y. P.; Thapa, R.; Acharya, G.; Lamsal, A., Study

on types of vaginitis and association between bacterial vaginosis and urinary tract

infection in pregnant women. IJBAR 2014, 5 (06), 305-7.

1. Joshi, S.; Subedi, Y. P.; Paudel, S. K., Antibacterial and antifungal activity of

heartwood of Acacia catechu of Nepal. J. Nepal Chem. Soc.2011, 27 (1), 94-99.

PRESENTATIONS

2. Subedi, Y. P.; Roberts, P.; Grilley, M.; Takemoto, J. Y.; Chang, C-W T.,

Amphiphilic kanamycin for treatment and diagnosis of the fungal infection. August

25, 2019, ACS annual meeting, San Diego, CA. (Poster)

1. Subedi, Y. P.; Alfindee, M. N.; Chang, C.-W. T. Fluorogenic probe for detection of

hydrogen sulfide in living system. November 29, 2016, SBC Annual Meeting, Logan,

UT. (Poster)