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DOUG A. MEDVETZ

THE SYNTHESIS, CHARACTERIZATION, AND ANTITUMOR PROPERTIES OF

Ag(I), Cu(II), AND Rh(III) METAL COMPLEXES

A Dissertation

Presented to

The Graduate faculty of the University of Akron

In Partial Fulfillment

of the Requirements for the Degree

Doctor of Philosophy

Doug A. Medvetz

August, 2008

THE SYNTHESIS, CHARACTERIZATION, AND ANTITUMOR PROPERTIES OF

Ag(I), Cu(II), AND Rh(III) METAL COMPLEXES

Doug A. Medvetz

Dissertation

Approved: Accepted:

Advisor Department Chair Dr. Wiley Youngs Dr. Kim Calvo

Co-Advisor Dean of the College Dr. Claire Tessier Dr. Ronald Levant

Committee Member Dean of the Graduate School Dr. Michael Taschner Dr. George Newkome

Committee Member Date Dr. Yang Yun

Committee Member Dr. Chris Ziegler

ABSTRACT

The anticancer drug cisplatin, which has been approved to treat ovarian, testicular, head and neck, certain types of lung, and metastatic breast cancers, has been a major advancement in cancer therapy. However, severe side effects including nausea, ear damage, vomiting, sensation loss in the extremities, and liver and kidney toxicity are usually experienced leading to the discontinuation of therapy. Along with the toxic side effects, resistance to this drug has become a major disadvantage. Due to these factors various other platinum derivatives have been explored leading to the discovery of carboplatin. Though carboplatin is quite active it still carries many of the same limitations as cisplatin, leading to the conclusion that the use of non-platinum metal complexes is essential. To date complexes of silver, rhodium, ruthenium, iron, copper, rhenium, titanium, and gold have shown promising results which may lead to the divergence from platinum based drugs.

This dissertation describes the synthesis, characterization, and anticancer properties of a variety of metal complexes. These include Ag(I) metal complexes along with a Cu(II) and a Rh(III) metal complex. Chapter 1 of this dissertation discusses a vast majority of the work that has been previously explored using non-platinum metal complexes as anticancer agents. Chapter 2 is devoted to the anticancer properties of three N-Heterocyclic carbene Ag(I) complexes. These complexes have shown anticancer

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properties in vitro against ovarian, breast, melanoma, colon carcinoma, and renal carcinoma cell lines with one of the complexes possessing preliminary in vivo anticancer properties against an ovarian cancer xenograft model. Chapter 3 is devoted to the synthesis, characterization, and in vitro anticancer properties of a Cu(II) thiaether complex and a Rh(III) thiaether complex against ovarian, breast, and lung cancer cell lines. Both complexes are active, however, the Cu(II) complex is more efficacious.

Chapter 4 describes the synthesis and characterization of a variety of new imidazolium salts, which are the precursors to N-Heterocyclic carbenes, and some of their respective

N-Heterocyclic carbene Ag(I) metal complexes that may have future use as anticancer agents.

DEDICATION

This dissertation is dedicated to all of the members of my immediate family that have been so supportive during the past few years.

ACKNOWLEDGEMENTS

First of all I would like to thank my advisor Dr. Wiley J. Youngs for all of his support and patience with me over the past five years. Without the enjoyable atmosphere that he allows his students to learn in this would not have been possible. To my co- advisor Dr. Tessier, I would like to say thank you for all of the knowledge that you passed down to me through your teaching in the classes I took and through our conversations in group meeting.

I would also like to thank all of the members of the Youngs group that I had the opportunity to interact with over the past five years Khadijah, Jered, Matt, Abdul, Carol,

Aysegul, Golf, Semih, Paul, Tammy, Jay, Sue, Mike, Nikki, Brian, and Amanda. A special thanks goes out to Jered Garrison and Matt Panzner for taking me under their wings when I first started.

A thanks goes out to Dr. Amy Milsted, Dr. Yang Yun, Dr. Joe Bauer, Dr. Daniel

Lindner, and Andrew Ditto for their invaluable help in cell culture. Without them the cancer project would not have made it.

I would also like to greatly thank my parents, Rick and Patti, for the patience and love they have expressed for me through all of the years of schooling I have gone through. They have always been behind every choice I have ever made and that has allowed me to get through this. Thanks, I love you.

A big thanks goes out to my siblings Steve, Jacki, and Amanda who have been there for me when I have needed to take the edge away from the stresses life in graduate school can bring. I love you guys.

Lastly, I would like to give a special thanks to my best friend and the love of my life. She knows who she is and she has made me into the man I have always wanted to be.

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TABLE OF CONTENTS

Page

LIST OF TABLES ...... xi

LIST OF FIGURES ...... xv

LIST OF SCHEMES ...... xx

LIST OF EQUATIONS ...... xxi

CHAPTER

I. INTRODUCTION TO METAL BASED ANTICANCER DRUGS ...... 1

1.1 Platinum Anticancer Drugs ...... 1

1.2 The Use of Silver as an Anticancer Agent ...... 4

1.3 The Use of Rhodium as an Anticancer Agent ...... 15

1.4 The Use of Ruthenium as an Anticancer Agent ...... 26

1.5 The Use of Iron as an Anticancer Agent ...... 33

1.6 The Use of Copper as an Anticancer Agent ...... 38

1.7 The Use of Rhenium as an Anticancer Agent ...... 43

1.8 The Use of Titanium as an Anticancer Agent ...... 46

1.9 The Use of Gold as an Anticancer Agent ...... 50

1.10 Conclusion ...... 53

II. ANTICANCER ACTIVITY OF Ag(I) N-HETEROCYCLIC CARBENE COMPLEXES DERIVED FROM 4,5-DICHLORO-1H-IMIDAZOLE ...... 55

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2.1 Introduction ...... 55

2.2 In Vitro Anticancer Activity ...... 56

2.3 In Vivo Anticancer Activity ...... 70

2.4 Conclusion ...... 72

2.5 Experimental ...... 72

III. SYNTHESIS, CHARACTERIZATION AND IN VITRO ANTICANCER ACTIVITY OF SOME THIAETHER TRANSITION METAL COMPLEXES ...... 77

3.1 Introduction ...... 77

3.2 Synthesis of 1-aza-4,7-thiacyclononane ...... 78

3.3 Synthesis of a Thiaether Rh(III) Metal Complex ...... 81

3.4 Synthesis of a Thiaether Cu(II) Metal Complex ...... 86

3.5 In Vitro Anticancer Activity ...... 88

3.6 Conclusion ...... 91

3.7 Experimental ...... 91

III. SYNTHESIS AND CHARACTERIZATION OF SOME Ag(I) N-HETEROCYCLIC CARBENES ...... 98

4.1 Introduction ...... 98

4.2 Synthesis of an Imidazolium Salt of Guanine ...... 99

4.3 Synthesis of an Imidazolium Salt of Guanosine ...... 104

4.4 Synthesis of 1-Carboxymethyl-3-methyl-4,5-dichloroimidazolium bromide ...... 108

4.5 Synthesis of 1,3-dimethyl-4,5-diiodoimidazolium iodide ...... 113

4.6 Synthesis of 1,3-dimethyl-4,5-diiodoimidazole silver acetate ...... 118

4.7 Synthesis of 1,3,5-Tris(1,3-dimethyl-4,5-dichloroimidazolium iodide)-2,4,6- trimethylbenzene ...... 123

4.8 Synthesis of 1,3,5-Tris(1,3-dimethyl-4,5-dichloroimidazole silver acetate)-2,4,6- trimethylbenzene ...... 128

4.9 Conclusion ...... 134

4.10 Experimental ...... 135

V. CONCLUSION ...... 141

BIBLIOGRAPHY ...... 144

APPENDICES ...... 151

APPENDIX A. CRYSTAL STRUCTURE DATA FOR C6H12Cl3NRhS2 (III-7) ..... 152

APPENDIX B. CRYSTAL STRUCTURE DATA FOR C6H13Cl2CuNS2 (III-8) ...... 157

APPENDIX C. CRYSTAL STRUCTURE DATA FOR C7H10BrN5O2 (IV-1) ...... 161

APPENDIX D. CRYSTAL STRUCTURE DATA FOR C12H18IN5O5 (IV-2) ...... 167

APPENDIX E. CRYSTAL STRUCTURE DATA FOR C6H7BrCl2N2O2 (IV-3) ...... 172

APPENDIX F. CRYSTAL STRUCTURE DATA FOR C5H7I3N2 (IV-4) ...... 176

APPENDIX G. CRYSTAL STRUCTURE DATA FOR C7H10I2N2O3 (IV-5) ...... 179

APPENDIX H. CRYSTAL STRUCTURE DATA FOR C24H27Br3Cl6N6O (IV-6)….182

APPENDIX I. CRYSTAL STRUCTURE DATA FOR C30H34Ag3Cl6N6O15 (IV-7).. 189

APPENDIX J. LIST OF ABBREVIATIONS ...... 198

APPENDIX K. APPROVAL FOR ANIMAL USE ...... 203

LIST OF TABLES

Table Page

1-1. Response Rates and Survival Time for Cancers Treated by Cisplatin...... 2

1-2. Toxicity of Cisplatin and its Analogues ...... 3

1-3. Anticancer Activity of 6-9...... 7

1-4. Cytotoxicity of 10-11 Compared to Cisplatin ...... 8

1-5. Cytotoxicity of Silver(I) Polymer Complexes...... 9

1-6. Cytotoxicity of Silver(I) Thioamide Polymers...... 11

1-7. Cytotoxicity of Silver(I) Coumarins...... 14

1-8. Cytotoxicity of Rh(II) Against KB Cells...... 17

1-9. Acute Mouse Toxicity and Cytotoxicity of Water Soluble Rh(II) Complexes ...... 20

1-10. Cytotoxicity of Rh(III) Complexes Against HCV29T Cells...... 26

1-11. Cytotoxicity of Ru(II) Arene Complexes Against A2780 Cells...... 30

1-12. Cytotoxicity of Bis(2-phenylazopyridine)ruthenium(II) Complexes...... 32

1-13. Cytotoxicity of Iron Semithiocarbazones Against SK-N-MC Cells ...... 36

1-14. Cytotoxicity of Fe(III) Porphyrin and Cisplatin Against Human Cancer Cells ...... 38

1-15. Cytotoxicity of Copper Carboxamidrazones Against B16F10 Cells...... 40

1-16. Cytotoxicity of Copper Carboxylate Against B16F10 Cells...... 42

1-17. Cytotoxicity of 66 Against Cancerous and Normal Cell Lines...... 45

1-18. Cytotoxicity of Titanocence Derivatives Against LLC-PK Cells ...... 49

1-19. Cytotoxicity of Au(III)-azpy Against Ovarian and Leukemic Cells ...... 53

2-1. Cytotoxicity of Ag(I) N-Heterocyclic Carbene Complexes...... 59

A-1. Crystal data and structure refinement for III-7 ...... 152

A-2. Atomic coordinates and equivalent isotropic displacement parameters for III-7.. 153

A-3. Bond lengths and angles for III-7...... 153

A-4. Anisotropic displacement parameters for III-7 ...... 156

A-5. Hydrogen coordinates and isotropic displacement parameters for III-7...... 156

B-1. Crystal data and structure refinement for III-8...... 157

B-2. Atomic coordinates and equivalent isotropic displacement parameters for III-8 .. 158

B-3. Bond lengths and angles for III-8...... 158

B-4. Anisotropic displacement parameters for III-8 ...... 159

B-5. Hydrogen coordinates and isotropic displacement parameters for III-8...... 160

C-1. Crystal data and structure refinement for IV-1...... 161

C-2. Atomic coordinates and equivalent isotropic displacement parameters for IV-1... 162

C-3. Bond lengths and angles for IV-1...... 163

C-4. Anisotropic displacement parameters for IV-1...... 165

C-5. Hydrogen coordinates and isotropic displacement parameters for IV-1...... 166

D-1. Crystal data and structure refinement for IV-2...... 167

D-2. Atomic coordinates and equivalent isotropic displacement parameters for IV-2 .. 168

D-3. Bond lengths and angles for IV-2...... 168

D-4. Anisotropic displacement parameters for IV-2 ...... 170

D-5. Hydrogen coordinates and isotropic displacement parameters for IV-2...... 171

E-1. Crystal data and structure refinement for IV-3...... 172

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E-2. Atomic coordinates and equivalent isotropic displacement parameters for IV-3.. 173

E-3. Bond lengths and angles for IV-3...... 173

E-4. Anisotropic displacement parameters for IV-3...... 174

E-5. Hydrogen coordinates and isotropic displacement parameters for IV-3...... 175

F-1. Crystal data and structure refinement for IV-4...... 176

F-2. Atomic coordinates and equivalent isotropic displacement parameters for IV-4.. 177

F-3. Bond lengths and angles for IV-4...... 177

F-4. Anisotropic displacement parameters for IV-4...... 178

F-5. Hydrogen coordinates and isotropic displacement parameters for IV-4...... 178

G-1. Crystal data and structure refinement for IV-5...... 179

G-2. Atomic coordinates and equivalent isotropic displacement parameters for IV-5. . 180

G-3. Bond lengths and angles for IV-5...... 180

G-4. Anisotropic displacement parameters for IV-5 ...... 181

G-5. Hydrogen coordinates and isotropic displacement parameters for IV-5...... 181

H-1. Crystal data and structure refinement for IV-6...... 182

H-2. Atomic coordinates and equivalent isotropic displacement parameters for IV-6 .. 183

H-3. Bond lengths and angles for IV-6...... 184

H-4. Anisotropic displacement parameters for IV-6 ...... 186

H-5. Hydrogen coordinates and isotropic displacement parameters for IV-6...... 188

I-1. Crystal data and structure refinement for IV-7...... 189

I-2. Atomic coordinates and equivalent isotropic displacement parameters for IV-7.... 190

I-3. Bond lengths and angles for IV-7...... 191

I-4. Anisotropic displacement parameters for IV-7...... 195

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I-5. Hydrogen coordinates and isotropic displacement parameters for IV-7...... 196

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LIST OF FIGURES

Figure Page

1-1 Molecular Structure of Cisplatin ...... 1

1-2. Molecular Structures of Cisplatin Analogues...... 3

1-3. Molecular Structures of Silver(I) Phosphines ...... 6

1-4. Molecular Structures of Silver(I) Carboxylate Polymers...... 9

1-5. Molecular Structure of the Thioamide 2-mercapto-3,4,5,6-tetra-hydropyrimidine .. 10

1-6. Molecular Structure of Silver(I) Thioamide Polymers...... 11

1-7. Molecular Structure of Coumarin...... 12

1-8. Molecular Structures of Silver(I) Coumarins ...... 13

1-9. General Structure of Rh(II) Carboxylate...... 15

1-10. Molecular Structures of Rh(II) Complexes Complexes ...... 17

1-11. Molecular Structure of Isonicotinic Acid ...... 18

1-12. Molecular Structures of Water Soluble Rh(II) Complexes ...... 19

1-13. Structure and Numbering for A) 9-ethylguanine and B) dinucleotide d(GpG) ...... 21

1-14. Crystal Structure of the 9-ethylguanine Complex of 23...... 22

1-15. Molecular Structure of the d(GpG) Complex of 23 ...... 23

1-16. Molecular Structure of 1-methyladenosine ...... 24

1-17. Molecular Structure of the 1-methyladenosine complex of 23 ...... 24

1-18. Molecular Structures of Rhodium(III) Complexes...... 25

1-19. Molecular Structures of Ru(III) Complexes of Imidazole and Indazole...... 27

1-20. Molecular Structure of NAMI-A...... 28

1-21. Molecular Structures of Ru(II) Arene Complexes ...... 29

1-22. Molecular Structures of Bis(2-phenylazopyridine)ruthenium(II) Complexes ...... 31

1-23. Molecular Structures of the Thiosemicarbazone Ligand Series...... 34

1-24. Molecular Structures of the Iron Thiosemicarbazone Complexes ...... 35

1-25. Cationic Portion of Porphyrin Ligand and its Fe(III) Metal Complex...... 37

1-26. Molecular Structure of A) appc and B) atpc...... 39

1-27. Molecular Structures of Copper Carboxamidrazones ...... 40

1-28. Molecular Structure of 5-amino-1-tolylimidazole-4-carboxylic acid ...... 41

1-29. Molecular Structure of Bis(5-amino-1-tolylimidazole-4-carboxylate)Cu(II) ...... 41

1-30. Molecular Structures of 6-amino-5-nitrosouracil Derivatives...... 43

1-31. Molecular Structures of Re(I) 6-amino-5-nitrosouracil Complexes...... 44

1-32. Molecular Structures of A) appt and B) [Re(CO)3(2-appt)Cl], 66...... 45

1-33. Molecular Structure of Titanocene...... 46

1-34. Molecular Structure of Titanocene X ...... 47

1-35. Molecular Structures of Bis(dipyridylphosphine)Au(I) Chloride Complexes ...... 51

1-36. Molecular Structure of Au(III)-azpy chloride salt...... 52

2-1. Molecular Structures of the Imidazolium Salts and Silver Acetate Complexes...... 57

2-2. Phase Contrast of OVCAR-3 Control ...... 60

2-3. Phase Contrast of OVCAR-3 Incubated with Cisplatin ...... 60

2-4. Phase Contrast of OVCAR-3 Incubated with II-4 ...... 60

2-5. Phase Contrast of OVCAR-3 Incubated with II-5 ...... 61

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2-6. Phase Contrast of OVCAR-3 Incubated with II-6 ...... 61

2-7. Phase Contrast of MB157 Control ...... 61

2-8. Phase Contrast of MB157 Incubated with Cisplatin ...... 62

2-9. Phase Contrast of MB157 Incubated with II-4 ...... 62

2-10. Phase Contrast of MB157 Incubated with II-5 ...... 62

2-11. Phase Contrast of MB157 Incubated with II-6 ...... 63

2-12. Live/Dead of OVCAR-3 Control ...... 65

2-13. Live/Dead of OVCAR-3 Incubated with Cisplatin ...... 65

2-14. Live/Dead of OVCAR-3 Incubated with II-4 ...... 65

2-15. Live/Dead of OVCAR-3 Incubated with II-5 ...... 66

2-16. Live/Dead of OVCAR-3 Incubated with II-6 ...... 66

2-17. Live/Dead of MB157 Control...... 66

2-18. Live/Dead of MB157 Incubated with Cisplatin ...... 67

2-19. Live/Dead of MB157 Incubated with II-4 ...... 67

2-20. Live/Dead of MB157 Incubated with II-5 ...... 67

2-21. Live/Dead of MB157 Incubated with II-6 ...... 68

2-22. Anticancer Activity of II-4 Against Melanoma ...... 69

2-23. Anticancer Activity of II-4 Against Renal Carcinoma ...... 69

2-24. Anticancer Activity of II-4 Against Colon Carcinoma...... 70

2-25. Necrotic Tumor Mass ...... 71

2-26. Normal Tumor Mass...... 71

2-27. View of Internal Organs…………………………………………………………...71

3-1. 1H NMR spectrum of III-6………………………………………………………….79

xvii

3-2. 13C NMR spectrum of III-6...... 80

3-3. X-ray crystal structure of III-7...... 82

3-4. 1H NMR spectrum of III-7...... 83

3-5. 13C NMR spectrum of III-7...... 84

3-6. X-ray crystal structure of (RhCl3[9]aneN2S)...... 86

3-7. X-ray crystal structure of III-8……………………………………………………...86

3-8. Molecular structure of (TACN)Cu(NO3)2…………………………………………..87

3-9. Anticancer Activity of Rh(III) and Cu(II) ...... 90

4-1. Molecular Structure of Guanine and Caffeine...... 100

4-2. X-ray crystal structure of IV-1...... 101

4-3. 1H NMR spectrum of IV-1 ...... 102

4-4. 13C NMR spectrum of IV-1...... 103

4-5. Molecular Structure of Guanosine...... 105

4-6. X-ray crystal structure of IV-2...... 106

4-7. 1H NMR spectrum of IV-2 ...... 107

4-8. Molecular Structure of 1-methyl-4,5-dichloroimidazole...... 109

4-9. X-ray crystal structure of IV-3...... 110

4-10. 1H NMR spectrum of IV-3 ...... 111

4-11. 13C NMR spectrum of IV-3...... 112

4-12. Molecular Structure of 1-methyl-4,5-diiodoimidazole...... 113

4-13. X-ray crystal structure of IV-4...... 115

4-14. 1H NMR spectrum of IV-4 ...... 116

4-15. 13C NMR spectrum of IV-4...... 117

xviii

4-16. Molecular Structure of 1,3-dimethyl-4,5-diiodoimidazole Silver Acetate...... 118

4-17. X-ray crystal structure of crystals isolated from IV-5 in H2O...... 120

4-18. 1H NMR spectrum of IV-5 ...... 121

4-19. 13C NMR spectrum of IV-5...... 122

4-20. Molecular Structure of 1,3,5-Tris(bromomethyl)-2,4,6-trimethylbenzene ...... 123

4-21. X-ray crystal structure of IV-6...... 125

4-22. 1H NMR spectrum of IV-6 ...... 126

4-23. 13C NMR spectrum of IV-6...... 127

4-24. X-ray crystal structure of IV-7...... 130

4-25. 1H NMR spectrum of IV-7 ...... 131

4-26. 13C NMR spectrum of IV-7...... 132

4-27. Packing diagram of the solid state structure of IV-7...... 134

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LIST OF SCHEMES

Scheme Page

1-1. Synthesis of Silver(I) Thioamide Polymers...... 10

1-2. Synthetic Route to New Titanocene Derivatives...... 48

3-1. Synthesis of 1-aza-4,7-thiacyclononane...... 78

LIST OF EQUATIONS

Equation Page

4-1...... 100

4-2 ...... 105

4-3...... 109

4-4 ...... 114

4-5 ...... 123

4-6 ...... 128

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CHAPTER I

INTRODUCTION TO METAL BASED ANTICANCER DRUGS

1.1 Platinum Anticancer Drugs

Since the discovery of its biological activity in 1965 by Rosenberg1 and FDA approval for general oncological treatment in 1978,2 cisplatin shown as 1 in Figure 1-1, has been used as a first-line chemotherapeutic agent for the treatment of many types of cancers. Cisplatin has been approved to treat ovarian, testicular, head and neck, certain types of lung, and metastatic breast cancers.3 This platinum drug has shown response rates between 70-90% in these types of tumors with survival rates ranging anywhere from 12-38 months depending on the cancer type and severity, as shown in Table 1-

1.4,5,6,7

H3N Cl Pt H3N Cl 1

Figure 1-1 Molecular Structure of Cisplatin

Table 1-1. Response Rates and Survival Time for Cancers Treated by Cisplatin Tumor Type Response Rate (%) Median Survival Rate (months)

Small Cell Lung Cancer 80-95 12-16

Ovarian (Stage II-IV) 70-80 26-38

Testicular (Stage I) 80-99 Most patients cured

Head and Neck 80-90 12-36

Non-small Cell Lung Cancer 20 12

The major drawback of using cisplatin is the side effects that are experienced by most patients. These side effects, in most cases, are extremely severe and lead to discontinuation of treatment. The most common symptoms are nausea, ear damage, vomiting, sensation loss in the hands, and renal (kidney) and hepato (liver) toxicity.

Because of these toxic side effects special dosing protocols where detoxifying agents, such as sodium dithiocarbamate, are administered simultaneously.8

Another major drawback of cisplatin use is the drug resistance that frequently occurs. There are various types of cancers that are inherently resistant to cisplatin, in which the drug has never had an effect. Along with inherent resistance, many types of cancers that are at one time sensitive to cisplatin as a first line treatment acquire resistance. This is a major problem in treating cancer patients and normally leads to reoccurrence of the tumor.8

Due to the toxicity and resistance factors associated with cisplatin, research into new platinum derivatives has lead to the development of carboplatin 2, nedaplatin 3, cycloplatam 4, and SKI 2053R 5.2,9 Of which only carboplatin is FDA approved. The

platinum drugs 2-5 are shown in Figure 1-2. These platinum drugs have shown efficacy and can be used in higher doses due to their lower reactivity in the body.9 These drugs are still mostly effective against the same class of cancers that cisplatin is active against, however they have shown efficacy against tumors that have acquired cisplatin resistance. Still, the same toxicity factors usually arise with the use of these new derivatives as shown in Table 1-2.2

O O H3N O H3N O Pt Pt H3N O H3N O 2 O 3

O O H2 OH O N O H2N O Pt Pt (H C) N O O 3 2 O H2 O 4 O 5

Figure 1-2. Molecular Structures of Cisplatin Analogues

Table 1-2. Toxicity of Cisplatin and its Analogues Drug Max Dose (mg/kg) Major Limiting Toxicity

Cisplatin (1) 3 Nephrotoxicitya

Carboplatin (2) 30 Nephrotoxictya

Nedaplatin (3) 3 Myelosuppressionb

Cycloplatam (4) 3 Myelosuppressionb

SKI 2053R (5) 10 Hepatotoxicityc a Severe toxicity to the kidneys b A condition where bone marrow activity decreases, resulting in fewer red blood cells, white blood cells, and platelets c Severe toxicity to the liver

Though the use of cisplatin and its derivatives have led to a major advancement in the treatment of cancer, the limitations due to toxicity and resistance has stimulated major research efforts into the discovery of non-platinum based chemotherapeutic agents for the treatment of cancer. Many other metals, including copper, gold, iron, rhenium, rhodium, ruthenium, silver, and titanium, have shown promising results when tested against numerous types of cancers.

1.2 The Use of Silver as an Anticancer Agent

Silver is a metal of interest in cancer therapy because its toxicity in humans is believed to be quite low.10 In fact, silver has been detected in 29 different human tissues in trace amounts.11 Though it is found in the body, there is no known physiological function for silver.11 There are already a variety of ways that silver is being used in medical applications, including coating of heart valves, cardiac catheters, and urinary catheters to reduce or prevent infection.12,13 The only major known side effect of silver ingestion is a permanent skin discoloration known as argyria. Argyria is not believed to be harmful to the body physically, and it takes an excessive consumption of silver to develop this condition.14

Research efforts have been focused on developing silver(I) complexes of a variety of different ligands to combat cancer. These include silver(I) complexes of phosphines, carboxylates, thio groups and thioamides, tripodal thioglycosides, and the natural product coumarin.15,16,17,18,19,20,21,22 These silver complexes show a high level of anticancer activity against a variety of different cell lines as will be discussed in the proceeding section .

The silver(I) phosphine complexes 6-9 have been explored by Berners-Price13 and 10-11 by McKeage.14 The molecular structures of these complexes are shown in

Figure 1-3. These silver phosphine complexes can be synthesized by the general route of adding two molar equivalents of the phosphine ligand to one equivalent of silver nitrate in either chloroform or acetone. They have been shown to be stable in light and are soluble in a variety of solvents. 31P NMR spectral studies show that these silver complexes are stable in solution for at least 24 hours and when nine molar equivalents of NaCl was added the spectra were unaffected indicating the chloride does not displace the silver ion from the ligands. Stability to NaCl solution is important because there is a high concentration of chloride in the bloodstream, so potential drugs need to be stable to chloride. Other 31P NMR spectral studies were done to determine the effect of glutathione (GSH) on these complexes. In the body, GSH is involved in many redox reactions including playing a major role in detoxification of harmful materials especially the chelation and removal of heavy metals. When twenty molar equivalents of GSH were added a white precipitate formed with all of the complexes except 9. This white precipitate was identified as the ligand of each respective complex indicating that

GSH can displace the silver in these complexes meaning that they are not stable to mimicked cellular conditions. In contrast complex 9 had no precipitate with GSH and the 31P NMR spectrum did not change indicating stability to GSH.

Ph2P PPh2 NO3 Et2P PEt2 NO3 NO3 Ag Ag Ph2P PPh2 Ag Ph2P PPh2 Et2P PEt2 Ph2P PPh2 6 7 8

R2P PR2 NO3 Ph2P PPh2 NO3 Ag Ag R2P PR2 Ph2P PPh2 10 R=(4-pyridyl) 9 11 R=(3-pyridyl) Figure 1-3. Molecular Structures of Silver(I) Phosphines

The antitumor activity of complexes 6-9 was determined in vitro against the B16 melanoma cell line, and in vivo against P388 leukemia and M5076 reticulum cell sarcoma in mice.13 The silver complex activity was compared to the respective free phosphine ligand. The results of these studies are shown in Table 1-3. IC50 values are given for the in vitro testing where IC50 represents the concentration needed to inhibit

50% of cell growth compared to untreated cells. Basically, the IC50 value represents at what concentration the compound allows only half of the normal growth rate of the cells when compared to the controls where no compound is used. The level of in vivo activity was determined by the percent increased lifespan (ILS) of the tested animals compared to the untreated animals where the silver complexes were administered on days 1-5 following the implantation of the tumors. The ILS represents how much longer the tested animals lived compared to the untreated animals. The doses given in this study were at the maximum dose tolerated (MTD) by the mice and are shown in Table 1-3.

The MTD was determined in a separate study and refers to the highest dose that could

be injected without causing animal death.13 The data indicate that the compounds that were tested in vivo were all relatively active.

Table 1-3. Anticancer Activity of 6-9 a Compound MTD P388 ILS M5076 ILS IC50

(µmol/kg/day) (%)b (%)b (µM)c

6 3 90 48 4

7 1 45 Not tested 4

9 5 50 Not tested Not tested

dpped 50 107 Not tested 60

depee 310 Inactive Not tested > 100

dppeyf 61 65 Not tested 25 a Maximally tolerated dose based on five daily injections in B6D2F mice b Compound considered active if greater than 40% for P388 and 25% in M5076 c Concentration in micromolar required for compound to inhibit 50% of cell growth after 2 h exposure d Free phosphine ligand of 6 e Free phosphine ligand of 7 f Free phosphine ligand of 9

The in vitro anticancer activity of 10-11 was explored against five different ovarian cancer cell lines, 41M and its cisplatin resistant subline 41M-cisR, CH1 and its cisplatin resistant subline CH1-cisR, and the cisplatin resistant cell line SKOV-3.14 The

41M-cisR cell line is cisplatin resistant due to decreased drug uptake, CH1-cisR is resistant due to increased repair capacity and tolerance of cisplatin-DNA adducts, and

SKOV-3 is resistant due to increased levels of glutathione.14 Cytotoxicity to normal cells was also assessed against primary rat hepatocytes (liver cells) to determine if 10-

11 are generally cytotoxic or if there is selectivity to cancerous cells. Table 1-4 shows

the results of these studies and compares the activity of the silver complexes to cisplatin. It appears that complexes 10 and 11 are active against all of the cell lines except SKOV-3, with IC50 values on the same order as cisplatin. Interestingly, these complexes also appear to be selective to the cancer cells.

Table 1-4. Cytotoxicity of 10-11 Compared to Cisplatin a b b b Compound IC50 IC50 IC50 IC50 IC50 IC50

Hepatocytes CH1b CH1-cisR 41Mb 41M-cisR SKOV-3

10 241 4 4 2 1.5 >100

11 - 2 3 2 2 >100

cisplatin - 0.1 1 0.5 1 5 a Concentrations in micromolar after 24 h exposure b Concentrations in micromolar after 96 h exposure

Silver(I) polymeric complexes have also been studied to determine the extent of their anticancer properties. Zhu has explored silver(I) carboxylates15 and Zachariadis has explored silver(I) polymeric complexes formed with the use of conjugated heterocyclic thioamides.17

The polymeric structures synthesized by Zhu have the structures [Ag(fbc)]n,

[Ag2(cpd)]n, and [Ag2(idc)]n where fbc is 4-fluorobenzoate, cpd is cyclopentane-1,1- dicarboxylate, and idc is iminodiacetate shown as 12-14 in Figure 1-4. These silver(I) extended networks are synthesized by dissolving Ag2O and the carboxylic acid, 1:2 for cpd and idc and 1:1 for fbc, in an ammonium solution. The solutions were allowed to sit in the air until a solid precipitate formed yielding the desired silver complexes which were confirmed by elemental analyses and IR spectroscopy.17

F O Ag O O O Ag O O Ag 13 O O Ag

O O Ag N 12 F O O Ag 14

Figure 1-4. Molecular Structures of Silver(I) Carboxylate Polymers

These silver polymers were tested in vitro against five solid carcinoma cell lines,

HeLa (cervical adenocarcinoma), HepG2 (hepatocellular carcinoma), BGC (gastric carcinoma), 95-D (lung carcinoma), CNE (rhinocarcinoma), and two normalized cell lines, NIH 3T3 (mouse fibroblast) and L-02 (human liver cells). The results of this data are summarized in Table 1-5. It appears that 12 was the only silver carboxylate polymer that is active and selective to the tumor cells. Complex 13 was not selective and 14 was not exceptionally active.

Table 1-5. Cytotoxicity of Silver(I) Polymer Complexesa Compound Hela HepG2 BGC 95-D CNE L-02 NIH3T3

12 8.1 8.7 9.3 8.7 16.2 17.4 41.6

13 4.2 4.2 6.3 4.2 10.8 4.2 4.2

14 20.6 13.4 25.4 26.8 60.1 25.8 11.7 a Data presented as IC50 concentration in micromolar after 48 h exposure.

The research explored by Zachariadis involves the self assembly of polymeric silver compounds using the thioamide 2-mercapto-3,4,5,6-tetra-hydropyrimidine, shown as 15 in Figure 1-5. Use of the thioamide was chosen because these types of ligands have a tendency to form bridged oligo and polynuclear compounds, a goal of this field of research. Two different silver polymers were synthesized using 15 as shown in

Scheme 1-1. The structures of these polymers are shown as 16 and 17 in Figure 1-6.

N S H 15

Figure 1-5. Molecular Structure of the Thioamide 2-mercapto-3,4,5,6-tetra- hydropyrimidine

Et3N 6AgBr + 6(15) {[Ag6(u2-Br)6(u2-15)4(u3-15)2]n} DMSO 16

Et3N 4AgNO + 6(15) {[Ag4(u2-15)6](NO3)4}n 3 DMSO 17

Scheme 1-1. Synthesis of Silver(I) Thioamide Polymers

NO3 NN N N S Ag Ag Br Ag S Br N N Ag S N S Br S Ag S N N N Br N Ag Ag N S N N 16 n 17 n

Figure 1-6. Molecular Structure of Silver(I) Thioamide Polymers

The in vitro anticancer activity of 16 and 17 was evaluated against the murine leukemia cell line L1210 and was compared to their cytotoxicity against the normal human T-lymphocyte cell lines Molt4/C8 and CEM. The results are shown in Table 1-

6. The ligand itself shows no cytotoxicity to any of the cell lines tested, however, the

Ag(I) complexes show good activity with some selectivity.

Table 1-6. Cytotoxicity of Silver(I) Thioamide Polymersa Compound L1210 Molt4/C8 CEM

15 >200 >200 >200

16 3.4 3.6 14

17 3.5 4.1 17 a Data is presented as IC50 values expressed in µg/mL.

The anticancer activity of silver(I) complexes of coumarins has been explored by Egan.19, 20 Coumarins are a large class of naturally occurring compounds that comprise 11

the general structure 18 shown in Figure 1-7. Naturally, they have been shown to possess minimal anticancer activity, however, when complexed to metals their activity greatly increases.

OH R 1 O R = OH or H R2 O O R 3 18

Figure 1-7. Molecular Structure of Coumarin

The silver(I) complexes of coumarins were synthesized by deprotonation of the respective coumarin derivative in an ethanolic solution of NaOH followed by the addition of silver nitrate. The silver complexes precipitated out of solution and were isolated without further purification and their structures were confirmed by IR and

NMR spectroscopy.23 Four different coumarin derivatives were used to synthesize the silver(I) complexes 6-hydroxycoumarin-3-carboxylatosilver 19, 7-hydroxycoumarin-3- carboxylatosilver 20, 8-hydroxycoumarin-3-carboxylatosilver 21, and coumarin-3- carboxylatosilver 22. The molecular structures of 19-22 are shown in Figure 1-8.

O O HO H O O Ag Ag H O O HO O O H 19 H 20

O O H H O O Ag Ag H O O H O O OH H 21 22

Figure 1-8. Molecular Structures of Silver(I) Coumarins

The anticancer activity of the coumarin ligands and their silver complexes was determined in vitro against the cancerous cell lines A-498 (human kidney adenocarcinoma) and Hep-G2 (human hepatocellular carcinoma), and compared to the normal cell lines HK-2 (human proximal tubular) and CHANG (human hepatic). The activity of 19-22 was compared to silver perchlorate and cisplatin and the results obtained from these studies are shown in Table 1-7. All of the silver complexes were as active as cisplatin against the cancerous cell lines (Hep-G2 and A-498), and showed better selectivity against the normalized cell lines (CHANG and HK-2).

Table 1-7. Cytotoxicity of Silver(I) Coumarinsa b Compound CHANG IC50 Hep-G2 IC50 HK-2 IC50 A-498 IC50

19 110 2.7 185 30

19-L >250 >250 >250 >250

20 >250 7.5 >250 35

20-L >250 >250 >250 >250

21 >250 5.5 >250 17

21-L >250 >250 >250 >250

22 170 6.5 >250 110

22-L >250 >250 >250 >250

AgClO4 19 7.6 65 44.4

Cisplatin 45 15 18 14 a IC50 concentrations are expressed in µM after 96 h exposure of silver complexes b The compounds labeled 19-L, 20-L, 21-L, and 22-L represent the coumarin free ligand of the respective silver complex.

The exact mechanism of how silver is active against the various cancer cells is not totally known, however, there have been a few reports that indicate possible answers to this question.22,24 The work in this area done by Egan using their coumarin silver complexes suggest that silver kills through an apoptotic pathway.22 Through electrophoresis studies they observed a DNA ladder pattern which is consistent with drugs causing apoptosis.22 Another indication that silver causes apoptosis is that the cells that undergo this type of death usually have increased levels of caspase 3 and 9.

Caspases are known as executioner proteins meaning they play a major role in cell death. 14

The cells that were treated with the silver coumarins had elevated levels of both caspases.22 The Egan group also explored if silver causes defects in the cell cycle.

From their studies they determined that silver does not allow cancer cells to enter G1

22 phase. In the cell cycle G1 phase is defined as the growth phase of the cycle where many biosynthetic pathways, including synthesis of the enzymes needed for DNA replication, resume.25 It is an important phase in cell growth and may have a major link to why silver is active in cancer cells.

1.3 The Use of Rhodium as an Anticancer Agent

Rhodium was discovered to have anticancer properties in the early 1970’s when

Bear and coworkers were exploring the activity of rhodium(II) carboxylates against various tumor cell lines.26 These structures are generally tetrabridged systems with short Rh-Rh bonds and contain various axial ligands as shown in Figure 1-9.

O O R

L O O Rh Rh O O L

R O O

Figure 1-9. General Structure of Rh(II) Carboxylate Complexes

Pruchnik27 and de Souza28 further explored rhodium(II) carboxylate derivatives for their anticancer activity. The work explored by Pruchnik involves not only rhodium(II) carboxylates, but also rhodium(II) complexes with the nitrogen containing ligands 2,2’-bipyridine and 1,10-phenanthroline. A major drawback of these types of rhodium complexes is their limited water solubility. Therefore, de Souza and coworkers explored the effect of axial ligand coordination on the solubility of these rhodium complexes.

The rhodium complexes 23, 24, and 25, shown in Figure 1-10, were synthesized by Pruchnik and showed anticancer activity against the human oral carcinoma KB cell line. These rhodium metal complexes were synthesized by the reaction of

Na4[Rh2(CO3)4] with acetic acid for 23, with formic acid and 2,2’-bipyridine for 24, and with formic acid and 1,10-phenanthroline for 25.29,30 The anticancer activity of these three rhodium(II) complexes is shown in Table 1-8. The ED50 value represents the concentration needed to cause 50% loss of total protein synthesis of the KB cells compared to control cells. This was determined according to the Lowry method where the amount of protein in control culture is compared to the amount of protein in the tested culture after a certain time period.31 Interestingly, 23, was almost four times as active as the two other derivatives in decreasing protein synthesis.

O O O O O H OH2 O Rh O Rh OH2 O O H O Rh N N = N O 2 Rh N N N H2O O O N N

23 24 H

O O H

OH O O 2 Rh Rh N N = N N N H2O N N N

Figure 1-10. Molecular Structures of Rh(II) Complexes

Table 1-8. Cytotoxicity of Rh(II) Against KB Cells a Rhodium(II) Complex ED50

23 30

24 100

25 100 a ED50 values are expressed in µM after 72 h incubation

De Souza and coworkers explored similar structures to Pruchnik, however they were interested in improving the water solubility of the dirhodium carboxylates by varying the axial ligands bound to the rhodium center. To enhance water solubility the

axially coordinated water ligands were replaced with isonicotinic acid.23 The structure of isonicotinic acid is shown in Figure 1-11.

O OH

Figure 1-11. Molecular Structure of Isonicotinic Acid

These new dirhodium carboxylates were synthesized by slowly adding two equivalents of isonicotinic acid in methanol to a stirred solution of the previously synthesized dirhodium carboxylates in methanol.25 The brownish-red products precipitated, were filtered, and washed with methanol to obtain the pure products 26-28 shown in Figure 1-12.

O O HO O HO O

O N O O O N O O Rh Rh Rh Rh O N O O N O O O

O O OH O O OH

26 27

HO O O

O N O O Rh Rh O O N O

O O OH

Figure 1-12. Molecular Structures of Water Soluble Rh(II) Complexes25

The Rh(II) complexes 26-28 were tested for their anticancer activity against

K562 cells, a human leukemia cell line and for their acute in vivo toxicity in male Balb- c mice. For the acute toxicity study the mice were broken into four groups of eight per complex. Each group was given an IP injection of a different dose to determine the

LD10 of each complex. The results of these studies are shown in Table 1-9. The LD10 values represent the dose that killed ten percent of the mice in the study.

As expected the LD10 directly correlates to the IC50 concentration with 28 being the most potent and toxic of the three.

Table 1-9. Acute Mouse Toxicity and Cytotoxicity of Water Soluble Rh(II) Complexes a Rh(II) Complex LD10 (µmol/kg) IC50 (µmol/L)

26 23.9 20.5

27 11.8 6.26

28 2.48 4.32 a IC50 concentrations after 24 h incubation

Because there are reports that dirhodium compounds inhibit DNA replication and protein synthesis similar to cisplatin,32, 33, 34 the mechanism by which these dirhodium carboxylates cause cell death has been extensively studied by Chifotides and

Dunbar.35, 36 For their initial studies they used the acetate structure 23. To determine how well these metal complexes bind to DNA they explored whether 23 could bind to the nucleobase 9-ethylguanine and the dinucleotide d(GpG). The structures and numbering schemes of 9-ethylguanine and d(GpG) are shown in Figure 1-13.

O 7 5 1 N 6 NH 8 N 4 2 9 N NH2 5' 3 O HO O 7 O 1 1' 7 N 5 4' 5 1 6 NH N 6 NH 8 3' 2' 8 9N 4 2 HO N 4 2 N NH2 9 N NH2 3 5' O P O 3 O A O 4' 1'

3' 2' HO B

Figure 1-13. Structure and Numbering for A) 9-ethylguanine and B) dinucleotide d(GpG)

The synthesis of the 9-ethylguanine complex of 23 was carried out by adding a slurry of 9-ethylguanine in water to a solution of 23 in water. The reaction was heated to 50 oC for 4 days yielding a green solution. The formation of the complex was monitored by 1H NMR spectroscopy. The structure of the complex was confirmed by

X-ray crystallography. The most notable feature in the 1H NMR spectrum is that the H8 proton shifts from 7.70 ppm in unbound 9-ethylguanine to 8.80 in the complex with 23.

The X-ray crystal structure shows that the guanine portion is bound to the rhodium atoms through an N7/O6 orientation as shown in Figure 1-14. Another interesting characteristic of this complex is that the guanine moiety is deprotonated at N1. This is important because the N1, N7, and O6 sites of guanine are all involved in the hydrogen bonding in DNA base pairing. This deprotonation may play a key role in the anticancer activity of these types of complexes by causing DNA pair mismatches eventually leading to cell death.

Figure 1-14. Crystal structure of the 9-ethylguanine complex of 23

The complex of 23 with d(GpG) was synthesized by adding 23 and d(GpG) to

o 1 D2O and stirring the solution at 37 C at pH 6. The H NMR spectrum was monitored until there was no presence of unbound d(GpG), resulting in a green solution. In the 1H

NMR spectrum the protons at the C8 position of the guanine ring for free d(GpG) appear at 7.71 and 8.00 ppm. These resonances disappear in the complex and two new ones arise at 8.80 and 8.58. This downfield shift is indicative of complexation of the rhodium metal center to the N7 of the guanine ring. The crystal structure was obtained and confirmed the formation of the complex shown in Figure 1-15.37

O O

OH O 2 Rh O Rh N H O O 2 N O O NN N O Bond angles and N N O P O NH2 lengths distorted for N O O clarity NH2

Figure 1-15. Molecular Structure of the d(GpG) complex of 23

It has also been shown that 23 can form complexes with the nucleoside derivative of adenine, 1-methyladenosine shown in Figure 1-16.30 This complex was synthesized similarly to the 9-ethylguanine derivative. The X-ray crystal structure of this complex was also obtained to verify how the 1-methyladenosine moieties were bound to the rhodium atoms. This complex indicates that these rhodium tetraacetate complexes have the ability to bind both adenine and guanine rendering it unnecessary for these complexes to rely on adjacent guanine molecules for DNA binding as cisplatin requires. The structure of the 1-methyladenosine complex is shown in Figure 1-17.

Again the N7 position of the adenine ring is bound to the rhodium atom and there is a stabilization of the complex by the amine group off of the six position through hydrogen bonding with the acetate group of the rhodium complex. Not only does this stabilize the complex, but it again has the possibility to interfere with normal DNA base pairing as seen in the 9-ethylguanine complex.

NH2 N N

N N O OH HO OH

Figure 1-16. Molecular Structure of 1-methyladenosine

N N NH OH 2 O O HO NN HO O O Rh O Rh O O O OH N N OH O O OH H2N N N

Figure 1-17. Molecular Structure of the 1-methyladenosine complex of 23

Rhodium(III) complexes have also been studied, though to a much lesser extent than rhodium(II), for their anticancer activity.38,39,40,41 The most promising rhodium(III) anticancer agents have been explored by Pruchnik where the ligands pyrazole, imidazole, triazine, and terpyridine have been used to complex rhodium(III).42 The rhodium(III) complexes were synthesized by the general procedure of adding an

42 ethanolic solution of the respective ligand to RhCl3 in ethanol. All of the products precipitated as the yellow solids 29-32. The molecular structures are shown in Figure

1-18.

HN HN N N N N Cl Cl Cl N N N H Rh H Rh Rh N Cl N Cl Cl Cl Cl N N Cl HN HN 30

N N N N N N Rh 3Cl N N N N N NH N Rh Cl Cl Cl 31 32

Figure 1-18. Molecular Structures of Rhodium(III) Complexes

These rhodium(III) complexes were tested for their in vitro anticancer activity against the human bladder cancer cell line HCV29T and compared to the activity of cisplatin. The results of this study are shown in Table 1-10. The ID50 represents the dose needed to inhibit 50% of cell proliferation. These results indicate that complexes

30 and 32 are the most active, with 32 showing about a 3-fold increase in activity over cisplatin. Complexes 30 and 32 may be more active due to their structures allowing for

DNA intercalation, but this has not been proven.

Table 1-10. Cytotoxicity of Rh(III) Complexes Against HCV29T Cells a Compound ID50 (µM)

29 102

30 6

31 67

32 0.7

cisplatin 2.4 a ID50 dose after 72 h incubation period

1.4 The Use of Ruthenium as an Anticancer Agent

The earliest reports of ruthenium complexes possessing anticancer activity came in the 1980’s when Clarke discovered the activity of fac-[Ru(III)Cl3(NH3)], however this drug has poor water solublity.43 To date there have been a variety of ruthenium complexes studied, however, only a few complexes have had activity comparable to cisplatin.43 The most promising class of ruthenium compounds are represented by

Ru(III) salts with imidazole and indazole ligands coordinated to the metal center through their respective free nitrogen atoms. Keppler was the first to report on these types of complexes shown as 33-34 in Figure 1-19.44 Impressively, 33, proved to be quite efficacious against colorectal carcinoma cells and many cisplatin resistant tumors with low toxic side effects.45 The most impressive characteristic of this complex is that it exceeded the activity of 5-fluoruracil, the standard treatment for colorectal cancer today.45 These results have led to 33 being explored in clinical trials.

So far, data from phase I and II have shown extremely promising results against liver, colon, head and neck, and endometrial cancers with active doses being well tolerated by patients.

N H NH N N Cl Cl Cl Cl HN HN Ru Ru HN Cl Cl Cl Cl N N HN HN

Figure 1-19. Molecular Structures of Ru(III) Complexes of Imidazole and Indazole

Another Ru(III) complex, 35 (also called NAMI-A), which is a derivative of 33 has shown very impressive anticancer and antimetastatic properties.46 This ruthenium complex, shown in Figure 1-20, was first synthesized because ruthenium complexes with coordinated dimethyl sulfoxide ligands have been shown to possess antitumor and antimetastatic properties.47 The remarkable preclinical activity of NAMI-A has led to its use in clinical trials and this potential drug has completed phase I as an antimetastatic agent.

O S H Cl Cl N Ru N Cl Cl H N

Figure 1-20. Molecular Structure of NAMI-A

More recently there has been work done exploring the activity of ruthenium(II) complexes. Sadler48 has explored ruthenium arene complexes and Reedijk49 has looked into ruthenium complexes of 2-phenylpyridine. Both of these types of complexes appear to be active against human ovarian carcinomas.

The work explored by Sadler involves Ru(II) complexes of the type [(η6-arene)-

Ru(A)(B)(C)] where the arene ligand is benzene or a substituted benzene, and A, B, and

C are monodentate ligands including halides, acetonitrile, and nicotinamide, or A-B is a bidentate ligand such as ethylenediamine or N-ethylethylenediamine. The arene ligands are believed to help in the stabilization and most of these complexes are ionic leading to enhanced water solubility. The metal complexes 36-45 were synthesized by the general

6 procedure of adding the respective [(η -arene)-Ru(A)]2, where A is a halide, to either acetonitrile, methanol, or benzene, and allowing them stir with NH4PF6, or adding in a diamine or isonicotinamide and letting them stir with NH4PF6. The molecular structures of these metal complexes are shown in Figure 1-21.

PF6 PF6 PF6

Ru NCCH3 Ru NCCH3 Ru NH2

Cl NCCH3 Br NCCH3 Cl H2N 36 37 38

PF PF6 PF6 6

Ru NH 2 Ru NH2 Ru NH2 I H N 2 Cl H2N I H2N 39 40 41

O PF PF6 6 PF6 O

Ru NH2 Ru NH Ru NH2

Cl H2N Cl H2N Cl H2N 43 42 44

PF6 O Ru N NH2 Cl Cl 45

Figure 1-21. Molecular Structures of Ru(II) Arene Complexes

The anticancer activity of complexes 36-45 was tested against the human ovarian cancer cell line A2780. The IC50 values were calculated after exposing the cells for 24 hours with each Ru(II) complex. The data from this study is shown in Table 1-

11. None of the complexes is as active as cisplatin, however, 42 and 44 are as active as

carboplatin. Complexes 36, 37, and 45 appear to have little to no activity. The mechanism by which these Ru(II) complexes kill cells has not fully been determined, but preliminary reports by Sadler indicate that they selectively bind to the N7 site of guanine.48

Table 1-11. Cytotoxicity of Ru(II) Arene Complexes Against A2780 Cells a Compound IC50

36 >150

37 >150

38 17

39 20

40 9

41 8

42 6

43 55

44 6

45 >150

carboplatin 6

cisplatin 0.5 a IC50 concentrations are expressed in µM after 24 h exposure

The Reedijk group has been working with the water soluble bis(2- phenylazopyridine)ruthenium(II) complexes to determine their level of anticancer

activity. These metal complexes can be readily synthesized by adding the ligand 1,1- cyclobutanedicarboxylic acid 46, oxalic acid 47, or malonic acid 48 to a stirring solution of α-[Ru-(azpy)2(NO3)2], where azpy is 2-phenylazopyridine, in acetone at 40 oC for four days. The molecular structures of these ruthenium complexes are shown in

Figure 1-22.

O O

O O N N O N O N O Ru N O Ru N

N N N N N N 46 47

O N N O O Ru N

N N N 48

Figure 1-22. Molecular Structures of Bis(2-phenylazopyridine)ruthenium(II) Complexes

The anticancer activity of these complexes was also tested against the human ovarian cancer cell line A2780 along with the cisplatin resistant version of this cell line

A2780cisR. The cells were incubated for 72 hours with the respective metal complexes and the IC50 values were then determined. The results from this study are shown in

Table 1-12. All three complexes were more active than carboplatin and on the order of

cisplatin against the normal cancer cell lines. Impressively, they were all more active than both cisplatin and carboplatin against the resistant cell lines.

Table 1-12. Cytotoxicity of Bis(2-phenylazopyridine)ruthenium(II) Complexesa Compound IC50 for A2780 IC50 for A2780cisR

46 7.2 4.9

47 6.3 6.3

48 7.9 6.2

cisplatin 2.3 7.8

carboplatin 8.2 41.6 a IC50 concentrations are presented in µM after 72 h incubation

The stability of these compounds was examined by 1H NMR spectroscopy under mimicked physiological conditions where the CDCl3 was modified to be at pH

7.4 by addition of phosphate buffer and contain a NaCl concentration of 0.1 M. These are the conditions of physiological saline solution and are the best mimic for in vivo bloodstream conditions. The spectra were monitored for one month to see what effects were caused. Metal complexes 47 and 48 showed no change in their respective spectra, however, 46 showed changes in its spectrum within five days, meaning 47 and 48 would probably be the most stable in vivo.

The mechanism by which ruthenium is active against cancerous cells is believed to occur through one of three different pathways. These three mechanisms include the formation of ruthenium-DNA interstrand crosslinks, ruthenium mimicking iron and

binding to transferrin, and lastly many rapidly dividing cells are hypoxic and have lowered pH which are favorable conditions for the binding of ruthenium to intracellular proteins.

Reedijk and co-workers have explored how ruthenium bound to guanine.50

They used the ruthenium compound mer-[Ru(terpy)Cl3] and reacted it with a variety of guanine derivatives and discovered that these types of ruthenium compounds formed interstrand DNA crosslinks with the N7 position of the guanine ring. This is interesting because it is opposite of the intrastand crosslinks formed by cisplatin.

Clarke and Keppler explored the ability of ruthenium to mimic iron.51,52

Because rapidly dividing cells require more iron they have an increased number of transferrin receptors on their surface. Work done in this area has shown that ruthenium binds to transferrin and therefore it is believed that transferrin carries ruthenium into the cell which may help explain its low toxicity in vivo.51

Clarke and Keppler have also explored why the hypoxia and lowered pH allow for Ru(III) complexes to be active in cells.51,52 They have shown that this is due to the ability of Ru(III) to readily undergo reduction to Ru(II) in these types of environments.

Therefore, the Ru(III) drugs are believed to be prodrugs and it is actually Ru(II) that is the active species. This mechanism is known as “activation by reduction.”51

1.5 The Use of Iron as an Anticancer Agent

There have been a variety of reports on the anticancer activity of iron chelated metal complexes.53,54,55,56 Recent work in this field has been explored by Richardson57 and Kawakami.58

The activity of iron complexes is believed to involve either in vivo redox chemistry or the targeting of superoxide dismutase.

The work done by Richardson et al. involves the chelation of Fe(II) and Fe(III) to 2-benzoyl and 3-nitrobenzoylpyridine thiosemicarbazones where the iron atom is wedged between two ligands. Four different thiosemicarbazone ligands, shown in

Figure 1-23, were used in this work to form the active Fe(II) and Fe(III) metal complexes.

NO2

N H H H H N N N N N N N N N H N N H N H S S S S N N N N

Figure 1-23. Molecular Structures of the Thiosemicarbazone Ligand Series

The iron thiosemicarbazone metal complexes were all synthesized by the general procedure of adding one equivalent of the appropriate ligand and Et3N to EtOH followed by the addition of 0.5 equivalents of Fe(ClO4)2 or Fe(ClO4)3. The reactions were refluxed under nitrogen for 30 minutes and upon cooling the green products precipitated. The Fe(II) derivatives 49-52 are shown in Figure 1-24. The Fe(III) complexes 53-56 comprise the same formula except the positive charge is counter- balanced by a perchlorate anion and are also shown in Figure 1-24.

N N N N N N H N S S N X N X Fe Fe N N S S H N N N N N N N

49 X= no anion 50 X= no anion 53 X= ClO4 54 X=ClO4

NO2

N N N N N H N H S X S X N N Fe Fe N N S S H N H N N N N N

O2N 51 X= no anion 52 X= no anion 55 X= ClO 56 X= ClO 4 4

Figure 1-24. Molecular Structures of the Iron Thiosemicarbazone Complexes

The anticancer activity of these iron complexes was tested in vitro against the

SK-N-MC neuroepithelioma cell line. The cells were incubated with the respective metal complex for 72 hours and the IC50 concentrations are reported in Table 1-13. All of the iron complexes were extremely active except for 56.

Table 1-13. Cytotoxicity of Iron Semithiocarbazones Against SK-N-MC Cells a Compound IC50 (µM)

49 0.21

50 0.21

51 0.21

52 0.12

53 0.15

54 0.35

55 0.17

56 >6.25 a Concentrations are reported after 72 h incubation with iron complex

The iron complex that was explored by Kawakami involves the complexation of

Fe(III) to the porphyrin 57 shown in Figure 1-25. This metal porphyrin complex was readily synthesized by the addition of 10 equivalents of FeCl3 to 57 in succinic acid

o - buffer at 80 C. The PF6 salt was precipitated by the addition of excess NH4PF6 and this salt was dissolved in acetone. The chloride salt 58, also shown in Figure 1-25, was then precipitated by the addition of tetraethylammonium chloride.

N N N N

N N

NH HN N Fe N

N N

57 58

Figure 1-25. Cationic Portion of Porphyrin Ligand and its Fe(III) Metal Complex

The Fe(III) porphyrin complex 58 and cisplatin were tested in vitro against the human lung cancer cell lines A549 and PC9, the human gastric cancer cell line MKN28, the human breast cancer cell line MCF7, the human colon cancer cell line SW948, and the human cervical cancer cell line HeLa. The results of these studies are presented in

Table 1-14. Impressively, the iron complex was more active than cisplatin against every cell line except the breast cancer cell line.

Table 1-14. Cytotoxicity of Fe(III) Porphyrin and Cisplatin Against Human Cancer Cells a a Cell Line IC50 58 IC50 cisplatin

A549 17.1 N/A

PC9 3.0 9.9

MKN28 1.8 12.6

SW948 6.8 9.1

Hela 4.3 5.8

MCF7 4.5 1.5 a IC50 concentrations presented in micromolar after 24 h incubation period

Though iron has been shown to kill cancer cells in vitro its use as an anticancer drug would appear not to be of utmost importance. This is due to the idea that many cancers require elevated levels of iron.59 It is believed that iron not only enhances tumor growth, but it also plays a major role as an initiator to tumorigenesis.59 It has been shown in mice that a diet high in iron increases colon cancer tumorigenesis.60

Therefore, it is believed that iron chelators may be better candidates as anticancer drugs than iron complexes.59

1.6 The Use of Copper as an Anticancer Agent

The use of copper for the treatment of cancer dates back to early 1980 with the report by Petering61 on the activity of copper thiosemicarbazones. More recent work has involved the use of copper complexes of carboxamidrazones62 and carboxylates.63

The carboxamidrazones are interesting because they have similar structures to the

thiosemicarbazones which were the first copper complexes to be reported for anticancer activity. Both classes of copper complexes have been explored by Padhye et al.62,63

The carboxamidrazone chemistry involves the use of the ligands 2- acetylpyridine-pyridine-2-carboxamidrazone (appc) and 2-acetylthiophene-pyridine-2- carboxamidrazone (atpc). These ligands are readily synthesized by refluxing pyridine-

2-carboamidrazide with excess 2-acetyl pyridine or 2-acetyl thiophene, respectively, in

EtOH for 2 hours. The molecular structures of appc and atpc are shown in Figure 1-26.

N N S N N N N

NH2 N NH2 A B

Figure 1-26. Molecular Structure of A) appc and B) atpc

The copper complexes of these ligands were also readily synthesized by adding equimolar amounts of appc or atpc and CuCl2 dihydrate to methanol and refluxing for 1 hour. The products precipitated out of solution as dark green crystalline material. The molecular structures of [Cu(appc)Cl2], 59, and [Cu(atpc)Cl2], 60, are shown in Figure 1-

27.

59 60

Figure 1-27. Molecular Structures of Copper Carboxamidrazones

The antiproliferative activity of copper complexes 59 and 60 was tested in vitro against the murine melanoma B16F10 cell line to determine if the different aryl substituents had any major effect on activity. The results of this study are presented in

Table 1-15. Surprisingly, the pyridine complex is almost two times more active than the thiophene complex suggesting that the aryl substituent may play a role in increasing anticancer activity.

Table 1-15. Cytotoxicity of Copper Carboxamidrazones Against B16F10 Cells a Complex IC50

59 6

60 10 a IC50 concentrations in micromolar after 24 h incubation period

The copper carboxylate chemistry involves the use of the ligand 5-amino-1- tolylimidazole-4-carboxylic acid, shown in Figure 1-28, which can readily be obtained by the alkaline hydrolysis of ethyl-5-amino-1-tolylimidazole-4-carboxylate. The copper complex was then synthesized by reacting two equivalents of 5-amino-1-tolylimidazole-

4-carboxylic acid with one equivalent of copper nitrate in methanol at pH 7. The product precipitates as the pure green solid 61 shown in Figure 1-29.

N N OH H N 2 O

Figure 1-28. Molecular Structure of 5-amino-1-tolylimidazole-4-carboxylic acid

Figure 1-29. Molecular Structure of Bis(5-amino-1-tolylimidazole-4-carboxylate)Cu(II)

The antiproliferative activity of 61 was also tested against the B16F10 cell line and compared to the activity of the carboxylic acid ligand and copper nitrate alone. The results for this study are presented in Table 1-16 and the complex is more active than either the ligand or copper nitrate alone.

Table 1-16. Cytotoxicity of Copper Carboxylate Against B16F10 Cells a Compound IC50

61 15

5-amino-1-tolylimidazole-4-carboxylic acid 50

Copper Nitrate >100 a IC50 concentration in micromolar after 24 h incubation period

There has been some recent work done trying to elucidate the mechanism of action of copper intracellularly. Zou and Somasundaram have explored reactive oxygen specie formation and DNA damage leading to p53 upregulation, repectively.64,65

It is believed that reactive oxygen species can induce oxidative stress in cells which leads to the initiation of apoptosis.66 These reactive oxygen species are believed to induce apoptosis by interacting with intracellular macromolecules, by attacking the cell membrane, and causing DNA strand breaks.64 The data obtained by Zou indicates that increased levels of copper in the cell, known as copper overload, become quite toxic and leads to the formation of reactive oxygen species which cause a decrease in glutathione activity, cellular redox state changes, and DNA damage leading to cell death.64

Somusandaram explored DNA damage caused by copper to a further extent.

They found that Cu(I) complexes cause cell cycle arrest leading to apoptosis.65 Their data indicate that the DNA damage caused by Cu(I) leads to the activation of the tumor suppressor gene p53 leading to apoptosis.65 The tumor suppressor p53 gets activated in cells under stress especially when DNA damage occurs.67

1.7 The Use of Rhenium as an Anticancer Agent

There have been a variety of reports on the activity of rhenium in the field of cancer therapy.68,69,70,71,72 More recently, work in this area has been explored by

Moreno-Carretero73 and Wong74 where both groups use Re(I) as the metal of choice.

The chemistry explored by Moreno-Carretero involves complexes of rhenium with 6-amino-5-nitrosouracil ligand derivatives. The uracil derivatives used in this chemistry are shown in Figure 1-30. The rhenium complexes can be readily synthesized by the equimolar reaction of the respective ligand and ReCl(CO)5 in

CH3CN at reflux for 3 hours. The structures of these complexes 62-65 are shown in

Figure 1-31.

O O O O NO H NO H NO H NO N N N N O N NH 2 O N NH2 O N NH2 H3CO N NH2 H

Figure 1-30. Molecular Structures of 6-amino-5-nitrosouracil Derivatives

CO CO CO CO OC CO OC CO OC CO OC CO Re Re Re Re O Cl O Cl O Cl O Cl N H N H N H N N O N O N O N O O N NH O N NH2 O N NH2 2 H3CO N NH2 H 62 63 64 65

Figure 1-31. Molecular Structures of Re(I) 6-amino-5-nitrosouracil Complexes

These rhenium complexes were tested against the human breast cancer cell lines

MCF7 and EVSA-T, the human neuroblastoma cell line NB69, the human glioma cell line H4, and the human bladder carcinoma cell line ECV. The IC50 values of these complexes were all around 10 µM for all cell lines tested indicating that there is little selectivity for the Re(I) 6-amino-5-nitrosouracil complexes.

The rhenium complexes explored by Wong involve complexation to the ligand

2-amino-4-phenylamino-6-(2-pyridyl)-1,3,5-triazine known as appt shown in Figure 1-

32. The Re(I) complex of this ligand was synthesized by the reaction of ReCl(CO)5 with appt in equimolar amounts in methanol for 24 hours forming the pure yellow solid

66 as shown in Figure 1-32.

CO N OC N Re N N OC N N Cl

H2N N N H2N N N H H A B

Figure 1-32. Molecular Structures of A) appt and B) [Re(CO)3(2-appt)Cl], 66

The anticancer properties of 66 were explored in vitro against the human epidermal carcinoma cell line KB-3-1 and its multi-drug resistance analogue KB-V-1, the human hepatocellular cancer cell line HepG2, the human cervical cancer cell line

HeLa, and the human lung fibroblasts cell line CCD-19Lu and compared to cisplatin.

The results of these studies are shown in Table 1-17. Interestingly, there is selectivity between the cancerous cell lines and the normalized lung fibroblasts, but 66 is not nearly as active as cisplatin.

Table 1-17. Cytotoxicity of 66 Against Cancerous and Normal Cell Linesa Compound KB-3-1 KB-V-1 HepG2 Hela CCD-19Lu

controlb >200 >200 >200 >200 >200

66 43.5 195 30.9 50.3 112

cisplatin 22.1 39.1 10.5 11.6 129 a IC50 concentrations in micromolar after 48 h incubation period b Control represents 4% DMSO in cell media

A recently published report by Baird on the mechanism of action of rhenium compounds against cancer cells in vitro elucidated some interesting results.75 They explored the ability of certain rhenium compounds to inhibit cathepsin B. Cathepsin B

is a protein that is believed to be elevated in tumors and is capable of degrading components in the extracellular matrix.75 In cancer cells, especially, it is believed to be involved in metastasis, angiogenesis, and tumor progression.76 The data collected by

Baird indicate that rhenium complexes have the ability to actively bind the active site of cathepsin B rendering it inactive indicating that this is, at least, one way that rhenium is active against cancer.

1.8 The Use of Titanium as an Anticancer Agent

The most important discovery in the field of titanium anticancer agents came with the introduction of titanocene.77 Titanocene, shown in Figure 1-33, reached clinical trials for the treatment of metastatic renal-cell carcinoma and metastatic breast cancer, however, it failed in Phase II due to unforeseen toxicity at clinically relevant doses.78 Recently there have been explorations into synthesizing titanocene derivatives to

decrease its lipophilicity and hopefully lower the in vivo toxicity. Quieroz79 and

Tacke80 have been involved in the more recent titanocene chemistry.

Cl Ti Cl

Figure 1-33. Molecular Structure of Titanocene

The chemistry being explored by Quieroz involves modification of the Cp rings to form the complex known as Titanocene X shown in Figure 1-34 as 67. This new titanium derivative can be readily synthesized by first synthesizing the substituted fulvene ligand and reacting it with titanium dichloride.

Me2N

H Cl Ti Cl Me2N H

Figure 1-34. Molecular Structure of Titanocene X

This new titanium dichloride derivative was tested in vivo against Ehrlich’s ascites tumor (EAT) inoculated mice. These mice were injected with 6 million cells

intraperitoneally and treated with doses of 2.5, 5.0, 10.0, 20.0, and 50.0 mg/kg for three consecutive days beginning 24 hours after cell injection. The treated mice were then compared to EAT mice that were given no drug and the mice were checked daily for survival rates. All of the untreated mice died within 21 days after being injected with the cancer cells. For the treated animals there was a dose-dependent survival rate. All of the animals that were given 2.5, 5, and 10 mg/kg doses also died within 21 days post cancer cell injection meaning that there was no effect of the drug at these doses.

However, the 47

animals given the 20 mg/kg dose survived for an average of 35 days post cancer cell injection and the mice given the 50 mg/kg dose survived for an average of 38 days.

This data indicates that this new derivative is quite effective at certain doses in vivo.

The work done by Tacke et al. involves modification of the Cp rings with dimethylamino and heteroaryl groups to try to improve the efficacy of this class of drugs. Modification of the Cp rings was explored in hopes of lowering the in vivo toxicity of these types of metallodrugs. These new titanium dichloride complexes were synthesized as shown in Scheme 1-2.

Scheme 1-2. Synthetic Route to New Titanocene Derivatives Li Li N N X THF o -78 C X X= S, O, N-CH3

Li X X N Cl

2 TiCl4 N N X THF 20h Ti

68 X=S 69 X=O 70 X=N-CH3

The anticancer activity of 68-70 was tested against the human carcinoma cell line LLC-PK to make a direct comparison to the activity of cisplatin and Titanocene X.

The IC50 values of this study are shown in Table 1-18. The data presented indicates

that, at least in vitro, these new titanocene derivatives are extremely active against carcinomas, and 70 is on the order of cisplatin. Interestingly, all three of these compounds are more active than Titanocene X which has already shown excellent efficacy in an in vivo mouse model.

Table 1-18. Cytotoxicity of Titanocence Derivatives Against LLC-PK Cells a Compound IC50 (µM)

68 240

69 28

70 5.5

Titanocene X 270

cisplatin 3.3 a IC50 values after 48 h incubation period

The mechanism of cancer activity for titanium is not completely understood, however, there have been some studies done to shed some light on this subject.81,82,83,84

It is believed that titanium complexes bind to nucleic acids and therefore suppress DNA or RNA synthesis.82,83 Other groups have shown titanium to inhibit protein kinase C which is an enzyme that regulates cell proliferation.81 Still others have demonstrated the ability of titanium to inhibit the enzyme topoisomerase II which plays an important role in DNA synthesis.84 From all of these studies an exact mechanism of action has not been determined, but may be determined in the future.

1.9 The Use of Gold as an Anticancer Agent

The use of gold in the field of treating cancer dates back to the late 1980’s with the work reported by Mirabelli.85 There have been some reports on the activity of Au(I) complexes86,87 and the most recent work in this area has been explored by Filipovska88 and Reedijk.89

The work studied by Filipovska involves the use of Au(I) complexes of phosphines, which have been previously shown to have promising anticancer effects.90

The previous work employed the use of diphenylphosphine ligands to complex the gold atom, whereas in the newer work dipyridylphosphines have been used as the ligands of choice. The change in ligand was a result of the in vivo hepatotoxicity that was found to occur with diphenylphosphine complexes in clinical development. The gold complexes

71-73, shown in Figure 1-35, were synthesized by the general procedure of reacting the respective phosphine ligand with tetrabutylammonium gold dichloride in methylene chloride yielding pure white solids.

Cl R P PR 2 Au 2

R2PPR2

N 71 R=

72 R=

N 73 R=

Figure 1-35. Molecular Structures of Bis(dipyridylphosphine)Au(I) Chloride Complexes

These gold complexes were tested against the human breast cancer cell line

MDA-MB-468 and the normalized breast cell line HMEC to determine their anticancer activity and selectivity between normal and cancerous cells. The major problem with all of the previously used gold compounds is that there was no selectivity, leading to toxicity problems.88 Complexes 72-73 showed no selectivity between the two cell lines tested, however 71 did. The IC50 for 71 against the cancer cell line was 1.6 µM and for the normal cell line was greater than 100 µM.

The work explored by Reedijk involves the use of Au(III) to complex the ligand

2-(phenylazo)pyridine known as azpy.89 These types of complexes also form gold salts, however, only one ligand is bound to the gold center. The gold azpy complex can be

synthesized by adding equimolar molar amounts of HAuCl4, LiCl, and azpy to methylene chloride with one drop of methanol at reflux for one hour. The molecular structure of this complex is shown as 74.

Cl Cl N Au N Cl N

Figure 1-36. Molecular Structure of Au(III)-azpy chloride salt

Complex 74 was tested against the human ovarian cancer cell lines A2780 and

A2780R (cisplatin resistant), and the murine lymphocytic leukemia cell lines L1210 and

L1210R (cisplatin resistant) and compared to cisplatin and HAuCl4. The data that resulted from these studies is presented in Table 1-19. Interestingly, 74 was in the range of cisplatin against the ovarian cancer cell lines but was not very efficacious against leukemia.

Table 1-19. Cytotoxicity of Au(III)-azpy Against Ovarian and Leukemic Cellsa Compound A2780 A2780R L1210 L1210R

HAuCl4 19.2 15.9 18.5 18.2

azpy 23.1 26.4 22.6 45.6

74 10.5 12.6 56.7 62.5

cisplatin 2.42 9.9 2.08 19.9 a IC50 concentrations in micromolar after 72 h incubation period

Though gold has been used clinically for 80 years its mechanism of action is not fully understood. However, the mechanism of action is believed to be related to the coordination at the gold center. It is believed that tetrahedral gold complexes attack the mitochondria of the cell. Studies have been done in rat hepatocytes with tetrahedral gold phosphines. The mitochondria were isolated and it was determined that much of the drug was uptaken due to its cationic and lipophilic nature. Once in the mitochondria it is believed that gold attacks the mitochondrial DNA producing crosslinks and strand breaks, along with inhibition of protein synthesis, efflux of calcium, and mitochondrial swelling. These effects eventually lead to the shutdown of the mitochondria and cell death.91

1.10 Conclusion

Due to the emergence of the platinum-based anticancer drugs into the clinical setting, research into the use of metal containing compounds as potential drugs has become a major area of research. Cisplatin, the first metal containing FDA approved

cancer drug, has been used to treat ovarian, testicular, head and neck, lung, and breast tumors as early as 1978. Though this drug has been approved, its use is becoming less frequent due to severe toxicity and resistance factors that will eventually render cisplatin obsolete. The new drug of choice, carboplatin, has shown some promising results to date, however, will eventually render the same fate as cisplatin as it too is a platinum-based drug.

Because of the shortcomings of the platinum-based drugs a plethora of potential drugs containing copper, gold, iron, rhenium, rhodium, ruthenium, silver, and titanium have been explored. All of these metals are active against various types of tumors, some in vitro and others in vivo. In fact, complexes of titanium and ruthenium have made it into clinical trials and have showed some promising results for future use. The goal of this area of research is to find new metals that are active in vivo and possess lowered toxicity and resistance factors when compared to platinum drugs. It appears that with the amount of quality data accumulated and the fact that some non-platinum drugs have entered clinical trials that this may be a reachable goal. The metals with the most promising chance of becoming clinically relevant, besides platinum, appear to be rhenium, rhodium, ruthenium, and silver due to their high level of activity.

CHAPTER II

ANTICANCER ACTIVITY OF Ag(I) N-HETEROCYCLIC CARBENE

COMPLEXES DERIVED FROM 4,5-DICHLORO-1H-IMIDAZOLE

2.1 Introduction

Recently, silver complexes have been reported to have anticancer activity in vitro.15,16,17,18,19,20,21,22 Egan has reported that silver complexes of coumarin derivatives possess anticancer activity against certain types of cancer.92 Zhu has reported that silver carboxylate dimers possess anticancer activity against human carcinoma cells.93

The use of silver as a chemotherapeutic agent in the treatment of cancer may lead to alternatives to presently used anticancer agents, especially the platinum-based cancer drugs. Silver is already being used in vivo to coat medical devices. Artificial heart valves, along with cardiac and urinary catheters, are already being coated with silver in medical applications to reduce or prevent the infection rate of various microbes.94,95,96,97

The toxicity of silver to the body is believed to be quite low. In fact, silver has been detected in 29 human tissues.98 Though, it is found in the body there is no known physiological function for silver. However, silver is metabolized in the body from insoluble silver salts, such as silver halides and phosphates, into soluble silver sulfide

albuminates that bind or form complexes with the amino and carboxyl groups in RNA,

DNA, and proteins.99 The problem with the silver sulfides is they cause a permanent discoloration of the skin known as argyria. Argyria is not believed to be harmful to the body physically, and it takes an excessive consumption of silver for this condition to occur.100

Based on the previously discussed reports of the antitumor activity of silver and our expertise in silver N-heterocyclic carbene complexes we examined the anticancer activity of Ag(I) complexes of N-heterocyclic carbenes.101,102,103,104 Herein is reported the anticancer activity of three Ag(I) N-heterocyclic carbene complexes derived from

4,5-dichloro-1H-imidazole.

2.2 In Vitro Anticancer Activity

1,3-dimethyl-4,5-dichloroimidazolium iodide II-1, 1-hexyl-3-methyl-4,5- dichloroimidazolium iodide II-2, 1-(2-napthylmethyl)-3-methyl-4,5- dichloroimidazolium iodide II-3, and their respective silver acetate complexes II-4, II-

5, and II-6 were synthesized by previously published procedures from our group.105,106

The molecular structures of the imidazolium salts, as well as, their silver acetate complexes are shown in Figure 2-1.

Cl N Cl N Cl N

N N Cl I Cl I Cl N I II-1 II-2 II-3

Cl N Cl N Cl N Ag O Ag O Ag O Cl N Cl N Cl N O O O II-4 II-5 II-6

Figure 2-1. Molecular Structures of the Imidazolium Salts and Silver Acetate Complexes

The MTT assay was run against the cancer cell lines HeLa S3 (cervical),

OVCAR-3 (ovarian), and MB157 (breast) to determine in vitro anticancer efficacy of the imidazolium salts II-1, II-2, and II-3, along with their silver respective silver complexes II-4, II-5, and II-6, silver nitrate and silver acetate. Cisplatin activity was also explored as a secondary comparison. These particular silver complexes were chosen because, at the time these studies were run, they were the most stable to light, water, and physiological sodium chloride solution. Also, these three complexes possess a range of differing alkyl groups off the 1-position of the imidazole ring making them interesting candidates to explore whether or not increased lipophilicity has any effect on anticancer activity. Cancer cells were plated at cell densities of 5,000 cells per well in

96-well plates and allowed to incubate overnight. The following day II-1, II-2, II-3, II-

4, II-5, and II-6, silver nitrate, silver acetate, and cisplatin were dissolved in DMSO and diluted

into the respective cell culture media to the desired micromolar concentrations. The dilution of the compounds in DMSO was used because it is a common protocol for in vitro cell line testing though platinum agents have been shown to have decreased activity when DMSO is involved.107 The media in the wells was replaced with fresh media containing test compound or DMSO for control. Cells were incubated for 72 hours. Following this test period MTT protocol was followed by adding 10 µL of MTT in PBS to each well and the plates were incubated for four hours. In viable, metabolically active cells, MTT is reduced in the mitochondria by the enzyme succinate dehydrogenase forming insoluble bluish purple formazan crystals. These formazan crystals are then resolublized by addition of 100 µL of SDS in dilute HCl. The optical density of each well is then read, spectrophotomically, at 570 nm by a microplate reader. LD50 concentrations, where LD50 stands for the concentration that causes a 50% reduction in cell viability, are reported in Table 2-1. The data show that II-4, II-5, and

II-6 are comparable in activity to cisplatin against the breast cancer cell line and on the order of cisplatin against ovarian cancer. The imidazolium salts showed little to no activity while silver nitrate and silver acetate did possess activity. Interestingly, the

Ag(I) complexes showed no activity against the cervical cancer cell line indicating some selectivity between cancers. Also, the trend of increasing drug lipophilicity equating to increased activity held true for the ovarian cancer. Unfortunately the same was not true for the breast cancer cell line where the least lipophilic drug II-4 was the most active of the silver complexes.

Table 2-1. Cytotoxicity of Ag(I) N-Heterocyclic Carbene Complexes85 HeLaa OVCAR-3a MB157a

Cisplatinb 12 12 25

AgNO3 50 35 5

AgOAc NA 20 12

II-4 >200 35 8

II-5 >200 30 20

II-6 >200 20 10 a LD50 concentrations are reported in micromolar b Cisplatin activity may be decreased due to use of DMSO NA = not achievable due to solubility of AgOAc

To determine the effect that silver complexes II-4, II-5, and II-6 and cisplatin had on the morphology of the OVCAR-3 and MB157 cells in culture, phase contrast pictures were taken of tested cell culture. The cells nuclei were dyed with Hoescht for clarity purposes. Briefly, cells were plated in 24-well plates and allowed to grow to confluency. Silver complexes II-4, II-5, and II-6 and cisplatin were dissolved in

DMSO and diluted into the respective cell culture media to the desired micromolar concentrations. The media in the wells was replaced with fresh media containing test compound or DMSO for control. Cells were incubated with test compounds at 50 µM for 36 hours. The results are shown as pictures in Figures 2-2 through 2-11.

Figure 2-2. Phase Contrast of OVCAR-3 control

Figure 2-3. Phase Contrast of OVCAR-3 incubated with cisplatin

Figure 2-4. Phase Contrast of OVCAR-3 incubated with II-4

Figure 2-5. Phase Contrast of OVCAR-3 incubated with II-5

Figure 2-6. Phase Contrast of OVCAR-3 incubated with II-6

Figure 2-7. Phase Contrast of MB157 control

Figure 2-8. Phase Contrast of MB157 incubated with cisplatin

Figure 2-9. Phase Contrast of MB157 incubated with II-4

Figure 2-10. Phase Contrast of MB157 incubated with II-5

Figure 2-11. Phase Contrast of MB157 incubated with II-6

The above pictures indicate that all the compounds tested have significant effect on the morphology of the cells. Changes in cell morphology are a direct result of loss of cell function, which is a necessity for anticancer agents. In terms of the silver complexes, while the test compounds were incubating with the cells, the plates were monitored at various time points to observe the morphological changes. Within 6-8 h the cells that were incubated with the silver complexes began to round up, shrink dramatically, and detach from the well surface indicating that the cells were undergoing apoptosis, or programmed cell death.108 These results are unlike necrosis which is usually characterized by loss of membrane integrity leading to an influx of extracellular fluids, cell swelling, and eventual cell lysis or autophagy which is the self digestion of the cells by lysosomes.109 The mechanism by which these silver complexes are active is underway and needs to be explored further because in apoptosis, DNA is also affected along with the activation of certain caspases.110

The live/dead cell assay was also run for II-4, II-5, and II-6 and cisplatin against the OVCAR-3 and MB157 cell lines to show cell death. Briefly, cells were

plated in 24-well plates and allowed to grow to confluency. Silver complexes II-4, II-

5, and II-6 and cisplatin were dissolved in DMSO and diluted into the respective cell culture media to the desired micromolar concentrations. The media in the wells was replaced with fresh media containing test compound or DMSO for control. Cells were incubated with test compounds at 50 µM for 36 hours. Live cells fluoresced red due to their metabolic activity that converted the stain C12-resazurin into fluorescent C12- resorufin. Dead cells loose their ability to maintain homeostasis, or normal cell function, and accumulate the Sytox green stain. This dye binds the cell nucleus and results in green fluorescence. Cells that dye yellow are in the process of undergoing cell death. The silver complexes II-4, II-5, and II-6 produced significantly higher death among the OVCAR-3 cell compared to cisplatin and control OVCAR-3 cells (p <

0.0001, n = 2). The viabilities of OVCAR-3 cells exposed to II-4, II-5, and II-6 was

11%, 0%, and 0%, respectively. Meanwhile, OVCAR-3 cells exposed to cisplatin resulted in 78% viability, which was not significantly different than the control cells

93% viability (p = 0.5579). All silver complexes and cisplatin produced significant death among MB157 breast cancer cells. The live/dead assay revealed 10% cell viability for MB157 cells exposed to cisplatin, which was significantly different than the 92% viability for the MB157 control cells (p<0.0001). Pictures were taken of the control and tested wells and are shown in Figures 2-12 through 2-21. Based on the live/dead assay, II-4, II-5, and II-6, showed significantly superior cell lysing capabilities with ovarian cancer compared to cisplatin. These silver complexes also were shown to completely kill the breast cancer cell line MB157.

Figure 2-12. Live/Dead of OVCAR-3 control

Figure 2-13. Live/Dead of OVCAR-3 incubated with cisplatin

Figure 2-14. Live/Dead of OVCAR-3 incubated with II-4 65

Figure 2-15. Live/Dead of OVCAR-3 incubated with II-5

Figure 2-16. Live/Dead of OVCAR-3 incubated with II-6

Figure 2-17. Live/Dead of MB157 control

Figure 2-18. Live/Dead of MB157 incubated with cisplatin

Figure 2-19. Live/Dead of MB157 incubated with II-4

Figure 2-20. Live/Dead of MB157 incubated with II-5

Figure 2-21. Live/Dead of MB157 incubated with II-6

Due to the relative ease of synthesis and availability of the compound the anticancer activity of II-4 was also explored against the human melanoma cell line

A375, the renal carcinoma cell line ACHN, and the colon carcinoma cell line HT1376.

This work was accomplished in collaboration with the Taussig Cancer Center at the

Cleveland Clinic’s Lerner Research Center using the SRB assay.111 The data that was obtained from these studies, which were run in triplicate, is presented in the following graphs. The IC50 concentrations for these cancer cell lines were calculated to be 20 µM for the melanoma and renal carcinoma lines, and 7 µM for the colon carcinoma.

Impressively, II-4 was as active as carboplatin against the cell lines tested. It would be expected that the other two Ag(I)-NHC complexes would also show similar activity, but this data was not obtained.

Antiproliferative effects of Metal-based agents on A375 Melanoma: 96 h growth

120

100 Cisplatin Carboplatin 80 II-4 60

0 Percent control growth +/- SEM +/- growth control Percent 0.512.5510152025

-20 uM

Figure 2-22. Anticancer Activity of II-4 Against Melanoma. Cells were incubated for 96 hours.

Antiproliferative effects of Metal-based agents on ACHN renal carcinoma: 96 h growth

120

100 Cisplatin Carboplatin 80 II-4 60

Percent control growth +/- SEM +/- growth control Percent 0.512.5510152025 -20 uM

Figure 2-23. Anticancer Activity of II-4 Against Renal Carcinoma. Cells were incubated for 96 h.

Antiproliferative effects of Metal-based agents on HT1376 colon carcinoma: 96 h growth

120

100 Cisplatin Carboplatin 80 II-4 60

Percent control growth +/- SEM +/- growth control Percent 0.512.5510152025 -20 uM

Figure 2-24. Anticancer Activity of II-4 Against Colon Carcinoma. Cells were incubated for 96 h.

2.3 In Vivo Anticancer Activity

Due to the high activity of these silver complexes, in vitro, a preliminary in vivo study was run to determine if II-4 was active against an ovarian cancer xenograft model. Briefly, ten million OVCAR-3 cells were injected subcutaneously into the back of three female athymic nude mice. Upon visible tumor growth, approximately six weeks, silver complex II-4 was injected subcutaneously at the tumor site every third day for ten days. Each dose consisted of 333 mg/kg of II-4 for a total of 1000 mg/kg over the ten day period. The mice were then necropsied to determine what effect II-4 had on the tumors, as well as, on the internal organs. Selected pictures of the tumors are shown in Figures 2-25 and 2-26. According to pathological results, II-4, caused major

necrosis of the tumor cells, however, showed no ill-effects to the major organs of the mice as shown in Figure 2-27.

Figure 2-25. Necrotic tumor mass

Figure 2-26. Normal tumor mass

Figure 2-27. View of Internal Organs

2.4 Conclusion

A new class of silver complex, the Ag(I) N-heterocyclic carbenes, was tested for its anticancer activity against ovarian, breast, cervical, melanoma, renal, and colon cancer cell lines. The activity of II-4, II-5, and II-6 was compared to the respective imidazolium salt precursors, along with silver nitrate, and silver acetate. The activity of cisplatin against these cancers was also used as a secondary comparison. By using the

MTT assay it was shown that all three silver complexes were most active against the ovarian and breast cancer cells, with little to no activity against the cervical cells.

Interestingly, a trend was seen in activity against the ovarian cell line where the activity increased from II-4 to II-6. This can be attributed to the increase in lipophilicity of the drugs leading to easier diffusion across the cell membrane. Phase contrast, Live/Dead, and SRB assays were also run to verify the activity of the silver complexes. A preliminary in vivo study was run for II-4 against an ovarian cancer xenograft model in athymic mice to explore the effect of these drugs at the tumor site in a living system.

The preliminary in vivo results indicate that silver complexes of this type will kill tumors in mice and need to be further pursued.

2.5 Experimental

Cell Lines. The human cancer cell lines OVCAR-3 and MB157 were purchased from

ATCC (Manassas, Virginia). The OVCAR-3 cell line was grown in RPMI 1640 media with 2 mM L-glutamine and modified to contain 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/L glucose, and 1.5 g/L sodium bicarbonate and was supplemented with

0.01 mg/mL bovine insulin and 20% fetal bovine serum. The cells were grown at 37 oC with 5% CO2 and passed every 2-3 days. The MB157 cell line was grown in DMEM with 4 mM L-glutamine modified to contain 4.5 g/L glucose and 1.5 g/L sodium bicarbonate and supplemented with 10% fetal bovine serum. The cells were grown at

o 37 C with 5% CO2 and passed every 2-3 days. The human cancer cell line HeLa was donated by Dr. Yang Yun of the Biomedical Engineering department at the University of Akron. The HeLa cell line was grown in DMEM/F-12K media supplemented with

1% antibiotic/antimicotic and 10% fetal bovine serum. The cells were grown at 37 oC with 5% CO2 and passed once a week.

MTT Assay. The MTT assay was purchased from Molecular Probes. The respective cell lines were grown to confluency and plated in 96-well plates at 5,000 cells per well in triplicate and allowed to incubate overnight. Compounds II-4, II-5, and II-6, cisplatin, silver acetate, and silver nitrate were dissolved in DMSO to a concentration of

0.1 M and diluted into cell culture media to the desired testing concentrations. Media in each well was removed and replaced with the fresh media containing test compounds.

The test compounds were allowed to incubate for 24, 48, or 72 hours after which the

MTT protocol was followed. A stock solution of MTT was prepared by adding 1 mL of

PBS to the pre-weighed vial containing MTT. A 10 µL aliquot of this stock solution was added to each well and allowed to incubate for 4 hours. A stock solution of sodium dodecyl sulfate was prepared by adding 10 mL of 0.01M HCl to the pre-weighed SDS vial. Following the 4 hour incubation period 100 µL of the SDS:HCl solution was added and incubated overnight. The optical density was read at 570 nm on Molecular

Devices Spectramax M2 plate reader.

Phase Contrast Study. Cells were plated at 50,000 cells per well in 24-well plates and the culture was allowed to grow to confluency. Compounds II-4, II-5, and II-6, and cisplatin were dissolved in DMSO to a concentration of 0.1 M and diluted into cell culture media to the desired testing concentrations. Media in each well was removed and replaced with the fresh media containing test compounds. The test compounds were incubated for 36 hours. Cells were rinsed with PBS and fixed for 10 minutes with freshly prepared 0.5% formaldehyde solution. After fixation, cells were rinsed and permeabilized with 0.2% Triton X solution for 10 minutes. Hoechst nuclear dye was prepared according to the manufacturer’s recommendations and carefully applied to the cells. After 30 minutes of incubation, cells were rinsed three times with PBS and visualized with fluorescence microscopy. Images were captured using AxioVision 200 by Zeiss with a 10x Plan Neofluor Zeiss objective, and a high resolution CCD HRm camera. The co-localized images were prepared using the AxioVision software version

4.6.

Live Dead Assay. The Live/Dead assay was purchased from Invitrogen. Cells were plated in 24-well plates. Compounds II-4, II-5, and II-6 and cisplatin were dissolved in

DMSO to a concentration of 0.1 M and diluted into cell culture media to the desired testing concentrations. Media in each well was removed and replaced with the fresh media containing test compounds. The test compounds were allowed to incubate for 36 hours. The Live/Dead stains were then prepared by dissolving 40 µg of C12-Resazurin in 100 µL of DMSO as a 1 mM stock solution. A 50 µM C12-Resazurin solution was then made up by diluting 2.5 µL of the stock solution into 47.5 µL of DMSO. The

Sytox Green solution was prepared by diluting 5 µL of a 10 µM stock solution in 45 µL of DMSO. The staining solution was prepared by diluting 48 µL of 50 µM C12-

Resazurin and 48 µL of Sytox Green stain into 4.704 mL of 1X PBS for final concentrations of 500 nM. The cell media was then removed from culture and the cells were washed with 1X PBS. Cells were then covered with 200 µL of Live/Dead staining solution and incubated for 15 minutes at room temperature protected from light. Lastly, the cells were visualized using an Axiovert 200 fluorescent microscope. Three random images were taken per well with an AxiocamHR using both fluorescein and rhodamine filters. The channels were combined and images were processed using Axiovision software. Cell viabilities were found to be significant using Anova with Tukey’s post test (α = 0.05).

SRB Assay. Experiments consist of two parts, one being the initial plates and one being the experimental plates. The initial plates have two columns with cells for control and are referred to as Aini. For the experimental plates cells are added to all wells and two columns are for control and referred to as Afin. Cells were plated in the morning at a density of 2000 cells per well in 96 well flat plates. The cells were allowed to adhere for six hours. II-4 was added to experimental plates and the initial plates were fixed with trichloroacetic acid (TCA). The initial plates were incubated in the refrigerator for at least one hour. Initial plates then washed with water and air dried. After 72-96 hour test period the experimental plates were fixed with TCA and incubated in the refrigerator for at least an hour. The experimental plates were washed with water and air dried. After dry 100 µL of Sulforhodamine-B (SRB, Sigma) dissolved in 1% acetic

acid was added to all plates. After 30 minutes Elute dye with 100 µL of 10 mM TRIS base was added. The plates were then read at 570 nm on a plate reader. Results were determined as percent control growth by the formula 100 * (Aexp - Aini)/(Afin – Aini).

Ovarian Cancer Xenograft Model. Three week old female athymic nude mice were purchased from Harlan and housed for one week prior to the experiment. Ten million cells were injected subcutaneously into the back of three animals, right below the ear.

The animals were monitored and the weights were recorded 2-3 times per week until the tumors grew to be visible (macroscopic tumors grew in about six weeks). The next day, and every third day following for ten days, the animals were injected with a dose of 333 mg/kg of compound II-4 subcutaneously at the tumor site for a total of 1000 mg/kg.

After ten days the animals were sacrificed and a full necropsy was performed.

CHAPTER III

SYNTHESIS, CHARACTERIZATION AND IN VITRO ANTICANCER ACTIVITY

OF SOME THIAETHER TRANSITION METAL COMPLEXES

3.1 Introduction

Research efforts into the development of new chemotherapeutic anticancer agents play an important role in the future treatment of cancer. The divergence from platinum based agents is becoming a point of emphasis with the goal being to find drugs that are as effective as platinum without the severity of side effects. Metallocene dichlorides and dirhodium carboxylates have shown anticancer activity, however neither class of compounds has demonstrated sufficient effectiveness to pursue past phase II clinical trials.112,113

Thiaether ligands have been used in transition metal complexation studies for many years.114,115,116 However, transition metal complexes of this type have not been extensively explored for their chemotherapeutic usage. These ligands appear to form relatively stable metal complexes, an attractive characteristic for in vivo use. Our work in this area was focused on using these ligands to complex Rh(III) and Cu(II) and test their activity against ovarian, breast, and lung cancer.

3.2 Synthesis of 1-aza-4,7-thiacyclononane

The thiaether ligand 1-aza-4,7-thiacyclononane, III-6, was synthesized similar to a published procedure as shown in Scheme 3-1.117 This thiaether ligand was used in forming the Rh(III) and Cu(II) complexes.

TsCl-Et3N LiBr HO N OH TsO N OTs O Br N Br H Ts Ts III-1 III-2 III-3 S EtOH H2N NH2 H Ts N N Phenol Br Br HS SH SS SS N HBr/CH3COOH DMF Ts III-6 III-5 III-4

Scheme 3-1. Synthesis of 1-aza-4,7-thiacyclononane

The 1H NMR spectrum of III-6, Figure 3-1, shows a singlet at 3.28 ppm for the

N-H proton and triplets at 3.03 ppm, 2.89 ppm, and 2.70 ppm for the sets of CH2 protons, and in the 13C NMR spectrum, shown in Figure 3-2, there were resonances at

33.1 ppm, 33.2 ppm, and 48.1 ppm all of which are consistent with published data. In the ESI-MS a peak at m/z of 163 was observed corresponding to the protonated form of

III-6.

-DMSO 6 d

in III-6 H NMRspectrum of 1

Figure 3-1.

3 in CDCl III-6 C NMR spectrum of 13

Figure 3-2.

3.3 Synthesis of a Thiaether Rh(III) Metal Complex

The RhCl3 complex of this thiaether ligand, III-7, was synthesized by dissolving

III-6 in ethanol and adding this solution into an equimolar solution of RhCl3 in ethanol.

Following the addition, an immediate dark orange precipitate formed and the solution was refluxed for 2 h. The precipitate was filtered, washed with cold ethanol, and dried with diethyl ether. In the 1H NMR spectrum of III-7, shown in Figure 3-4, there is a singlet at 2.99 ppm for the N-H proton, and multiplets at 3.10 ppm, 3.31 ppm, 3.54 ppm, and 3.83 ppm. The multiplets arise from the coordination of the rhodium atom to the ring system. Due to rhodium coordination the protons situated closer to the rhodium center now differ from the protons pointed away from the rhodium center. Along with the differing protons, the rhodium atom itself causes splitting in the 1H spectrum leading to the array of multiplets. In the 13C NMR spectrum, shown in Figure 3-5, there are two resonances at 36.0 ppm and 36.5 ppm for the two carbons of the ethylene linkages between the sulfur atoms and a resonance at 52.1 ppm for the carbons nearest to the nitrogen atom. For the ESI-MS a peak at m/z of 373 appears representing the protonated form of the complex. The decomposition point of this metal complex was determined to be 265 oC and crystals suitable for X-ray diffraction of III-7 were grown from a concentrated saline solution by slow evaporation. The solid state structure is shown in Figure 3-3.

Figure 3-3. X-ray crystal structure of III-7. Thermal ellipsoids are shown at 50% probability. Hydrogen atoms were removed for clarity.

O 2 in D III-7 H NMR spectrum of 1

Figure 3-4.

-DMSO 6 d

in III-7 C NMR spectrum of 13

Figure 3-5.

The RhCl3 thiaether complex, III-7, bears resemblance to the structure

[RhCl3([9]aneN2S] that was synthesized and crystallographically characterized by

118 Mattes. The molecular structure of [RhCl3([9]aneN2S] is depicted in Figure 3-6. The major difference, structurally, between the two compounds is that III-7, possesses two sulfur atoms and one nitrogen in the ring system, while [RhCl3([9]aneN2S] possesses two nitrogen atoms and one sulfur. For III-7, the S(1A)-Rh(1) and S(1B)-Rh(1) bond distances are 2.19(1) Å and 2.32(1) Å compared to 2.24(2) Å for the S(1)-Rh(1) distance of [RhCl3([9]aneN2S]. The N(1)-Rh(1) distance of III-7 is 2.06(1) Å while the

N(1)-Rh(1) and N(2)-Rh(1) distances for [RhCl3([9]aneN2S] are 2.03(3) Å and 2.04(3)

Å, respectively. The Cl-Rh-Cl bond angles for III-7 are on average 92.16(16) Å and for

[RhCl3([9]aneN2S] are on average 91.78(10) Å.

Figure 3-6. Molecular Structure of [RhCl3([9]aneN2S]

3.4 Synthesis of a Thiaether Cu(II) Metal Complex

The CuCl2 thiaether complex, III-8, was synthesized by adding a solution of

CuCl2 in ethanol to a stirring solution of III-6 in ethanol at room temperature. An immediate green precipitate formed and the solution was stirred overnight at room temperature. The green precipitate was filtered and washed with ethanol. The NMR spectrum of III-8 showed one broad peak due to the paramagnetic Cu(II) metal center.

In the ESI-MS spectrum a peak arises at m/z of 261 which represents III-8 with the loss of one chloride. The decomposition point of this metal complex was determined to be

147-155 oC and crystals suitable for X-ray diffraction of III-8 were grown from a concentrated solution of CH3CN by slow evaporation. The solid state structure is shown in Figure 3-7.

Figure 3-7. X-ray crystal structure of III-8. Thermal ellipsoids are shown at 50% probability. Hydrogen atoms were removed for clarity.

The copper(II) complex III-8 has some interesting properties in terms of its solid state structure. These properties are similar to other closely related copper(II) complexes of triazacyclononane (TACN) that have been recently synthesized and characterized by X-ray crystallography.119 In terms of electronics copper(II) is a d9 metal which means that it should experience strong Jahn-Teller distortion. This distortion helps to lower the overall energy of non-linear degenerate molecules such as copper(II) complexes by elongation of bonds in the complex. The most notable bond distortions in the solid state structure of III-8 are the two S-Cu bonds. The S(1)-Cu(1) bond distance is 2.34(1) Å and the S(2)-Cu(1) bond distance is 2.55(1) Å. These distances are obviously not within standard deviation of one another and indicative of the Jahn Teller effect. The (TACN)Cu(NO3)2 complex, shown as Figure 3-8, also experiences this distortion. The N(X)-Cu(1) bond lengths, where X is 1, 2, and 3, are

2.22(1) Å, 1.99(2) Å, and 2.03(1) Å, respectively indicating, as expected, bond

elongation. The

N(1)-Cu(1)

distance of III-8

is 2.07(1) Å.

Figure 3-8. Molecular structure of (TACN)Cu(NO3)2 87

Interestingly, there are also some distinct comparisons between III-7 and III-8.

The N(1)-Rh(1) distance of 2.06(1) Å is very comparable to the N(1)-Cu(1) distance of

2.07(3) Å. It also appears that in both complexes there is a shorter S-Rh distance and a longer S-Rh distance for the two sulfur atoms in the ring system. The shorter bond lengths are 2.19(1) Å and 2.34(1) Å for the rhodium and copper complexes, respectively, and the longer distances are 2.32(1) Å and 2.55(1) Å, respectively. The

Rh-Cl bond distances are on average 2.41(2) Å while the Cu-Cl distances average approximately 2.29(2) Å.

3.5 In Vitro Anticancer Activity

The in vitro anticancer activity of metal complexes III-7 and III-8 was explored using the MTT assay against the human cancer cell lines OVCAR-3 (ovarian), MB157

(breast), and A549 (lung). Cancer cells were plated at cell densities of 5,000 cells per well in 96-well plates and allowed to incubate overnight. The following day complexes

III-7 and III-8 were dissolved in DMSO and diluted into the respective cell culture media to the concentration of 50 µM. The media in the wells was replaced with fresh media containing test compound or DMSO for control. Cells were incubated for 24 h.

Following this test period MTT protocol was followed by adding 10 µL of MTT in PBS to each well and the plates were incubated for four hours. In viable, metabolically active cells, MTT is reduced in the mitochondria by the enzyme succinate dehydrogenase forming insoluble bluish purple formazan crystals. These formazan crystals are then resolublized by addition of 100 µL of SDS in dilute HCl. The optical

density of each well is then read, spectrophotomically, at 570 nm by a microplate reader. The results of these studies are shown in Figure 3-9. As shown in the following graph the Rh(III) complex is only minimally active against the three tested cell lines, however, the Cu(II) complex appears to be quite active against all three cell lines when compared to the control wells where 0.5% DMSO in cell culture media was added.

This activity difference may be attributed to the fact that Rh(III) forms very stable complexes and longer incubation times may be necessary to see the full potential of this anticancer agent. Though the copper complex shows excellent in vitro activity, preliminary in vivo toxicity studies showed this complex to be extremely toxic even at very low doses making its use as an anticancer agent impractical.

Anticancer Activity of Rh(III) and Cu(II) 100

OVCAR-3 80

MB157 70

A549 60

40 Percent Viability 30

0 control 7 8 Metal Complex

Figure 3-9. Anticancer Activity of Rh(III), 7, and Cu(II), 8. Data is presented as the percent viability of cells compared to the control cells where only DMSO in cell culture media was administered. Cells were tested for 24 h.

In terms of in vitro anticancer activity the thiaether Cu(II) complex appears to be comparable to the activity of the Ag(I)-NHC complexes that were discussed in the previous chapter. The silver complexes are slightly more active against the ovarian cancer and the breast cancer lines showing inhibition of 90-100% of the cells at 50 µM compared to 70-80% inhibition for the copper complex. On the other hand, the Cu(II) complex shows increased activity against the lung cancer cell line inhibiting 70% of the cells and the silver complexes range from 25-50% inhibition. The Rh(III) complex is about 8-10 fold less active than the Ag(I)-NHC complexes against the ovarian and

breast cancer cells, and against the lung cancer cells the rhodium complex is quite inactive.

3.6 Conclusion

In this chapter the synthesis, characterization, and in vitro anticancer activity of a

Rh(III) and a Cu(II) thiaether complex are described. The Rh(III) thiaether complex shows structural similarities to the previously synthesized rhodium trichloride complex

1 13 [RhCl3([9]aneN2S]. The X-ray crystal structure, H, and C NMR spectra are described in detail for the rhodium complex. The Cu(II) complex described in this chapter displays some interesting structural properties in comparison to similarly synthesized TACN derivatives. The X-ray crystal structure of this copper complex is described which details the strong Jahn Teller effect that is experienced by this d9 metal complex. Lastly, these two thiaether complexes were tested for their in vitro anticancer properties against ovarian, breast, and lung cancer cell lines. In comparison, the Cu(II) complex is approximately three-fold more active against all three cell lines.

3.7 Experimental

General. The starting materials diethanolamine and copper dichloride were purchased from Alfa Aesar. Thiourea was purchased from Acros Chemical and rhodium trichloride was purchased from Strem Chemical. Standard grade silica gel with porosity of 60 Å, particle size of 60-63 µm, density of 0.4 g/mL, and pH of 6.5-7.5 was purchased from Sorbent Technologies. All chemicals were used without further

purification. 1H NMR data was obtained using either a Varian 300 or 400 MHz instrument. 13C NMR data was obtained using a Varian 300 MHz instrument. The spectra were referenced to the residual protons and the 13C signals of the deuterated solvents. Mass spectrometry data was collected from either the mass spectrometry laboratory at the University of Akron or at the Mass Spectrometry and Proteomics

Laboratory at The Ohio State University. Elemental analysis data was obtained from the University of Illinois, Urbana-Champaign. Melting points were taken on a Thomas

Scientific Capillary Melting Point Apparatus.

X-ray crystallography. Crystals of III-7 and III-8 were coated in paratone oil, mounted on a CryoLoop™ and placed on a goniometer under a stream of nitrogen. X- ray data were collected using a Bruker Apex CCD diffractometer with graphite- monochromated Mo Kα radiation (λ= 0.71073 Å). The data was integrated using

SAINT. An empirical absorption correction and other corrections were applied using multi-scan SADABS. A Bruker SHELXTL package was used for the structure solution, refinement and modeling of the crystals. The structures were determined by full-matrix least-squares refinement of F2 and the selection of appropriate atoms from the generated difference map.

N,N-bis[2-(p-tolylsulfonyloxy)]-toluene-p-sulfonamide (III-2). Diethanolamine III-1

(20.50 g, 0.195 mol) was dissolved in triethylamine (300 mL) in an ice bath under nitrogen atmosphere. To this solution was added p-toluenesulfonyl chloride (102.0 g,

0.536 mol) over a period of 15 minutes. The ice bath was removed and the cloudy solution was stirred at RT overnight. The excess triethylamine was decanted and the

sticky brown solid was washed with water until it could be filtered. The solid was then recrystallized twice with an ethanol-toluene (5:1) mixture yielding an off-white

1 crystalline solid (74.61 g, 67%). H NMR (300 MHz, d6-DMSO) δ 2.44 (s, 3H), 2.47 (s,

3H), 3.38 (t, 4H), 4.12 (t, 4H), 7.30 (d, 2H), 7.37 (d, 2H), 7.62 (d, 2H), 7.77 (d, 2H). 13C

NMR (75 MHz, d6-DMSO) δ 21.7, 21.8, 48.6, 68.4, 127.3, 128.1, 130.1, 130.2, 132.4,

135.3, 144.3, 145.4. m.p. 98-99oC.

N-tosyl-bis(2-bromoethyl)-amine (III-3). Compound III-2 (74.61 g, 0.131 mol) was dissolved in 250 mL of acetone and LiBr (30.24 g, 0.348 mol) was added. The solution was refluxed under nitrogen atmosphere overnight. The reaction mixture was cooled to

RT yielding a tan precipitate that was filtered and washed with acetone. The solvent of the filtrate was removed in vacuo resulting in a brown powdery solid. The solid was added to 50 mL of CH2Cl2 and the insoluble portion was filtered off. The solvent was

1 removed in vacuo yielding a light brown solid (36.92 g, 60%). H NMR (300 MHz, d6-

13 DMSO) δ 2.45 (s, 3H), 3.52 (s, 8H), 7.35 (d, 2H), 7.73 (d, 2H). C NMR (75 MHz, d6-

DMSO) δ 21.8, 29.8, 51.7, 127.4, 130.3, 144.4. m.p. 63oC

N-tosyl-bis(2-mercaptoethyl)-amine (III-3a). Compound III-3 (36.92 g, 0.096 mol) was added to a solution of thiourea (19.86 g, 0.261 mol) in de-aerated EtOH. The solution was refluxed overnight yielding a white solid that was filtered and washed with

1 Et2O and air dried (40.56 g, 63%). H NMR (300 MHz, d6-DMSO) δ 2.43 (s, 3H), 3.36

13 (t, 2H), 3.55 (t, 2H), 7.46 (d, 2H), 7.75 (d, 2H). C NMR (75 MHz, d6-DMSO) δ 20.9,

30.0, 47.2, 127.4, 130.5, 146.1. m.p. 174oC

N-tosyl-bis-(2-thioethyl)-amine (III-4). KOH (36.10 g, 0.658 mol) was added to 300

mL of de-aerated water and compound III-3a (36.92 g, 0.068 mol) was added slowly over a 15 minute period. The solution was refluxed for 3 h after which was allowed to cool to RT and filtered through celite. Concentrated HCl saturated with N2 was added dropwise until pH~3 and the product was extracted with CH2Cl2 (5 x 100 mL). The combined organic layers were washed with water and dried over anhydrous Na2SO4 and the solvent removed in vacuo yielding a white solid (16.75 g, 84%). 1H NMR (300

MHz, d6-DMSO) δ 1.43 (t, 2H), 2.44 (s, 3H), 2.73 (q, 4H), 3.29 (t, 4H), 7.33 (d, 2H),

13 7.71 (d, 2H). C NMR (75 MHz, d6-DMSO) δ 21.6, 24.1, 52.9, 127.2, 130.0, 143.9.

1-tosyl-1-aza-4,7-dithiacyclononane (III-5). Cesium carbonate (12.89 g, 0.039 mol) was added to 500 mL of dry DMF under N2. Compound III-4 (10.42 g, 0.036 mol) and

1,2-dibromoethane (3.0 mL, 0.036 mol) were added to 300 mL of dry DMF under N2 in an addition funnel. The cesium carbonate solution was heated to 55 oC and addition began dropwise for 24 h, followed by continual stirring at 55 oC for 24 h. The solution was then cooled to RT and the solvent removed in vacuo. The white solid was added to

100 mL of CH2Cl2 and washed with 200 mL of water. The organic layer was dried and the solvent removed yielding a sticky yellow solid. The pure product was isolated through column chromatography with CH2Cl2 as the solvent (5.34 g, 47%). Rf = 0.43

1 [silica gel: CH2Cl2]. H NMR (300 MHz, d6-DMSO) δ 2.44 (s, 3H), 3.14 (d, 4H), 3.17

13 (s, 4H), 3.39 (t, 4H), 7.34 (d, 2H), 7.70 (d, 2H). C NMR (75 MHz, d6-DMSO) δ 21.6,

32.5, 34.3, 53.6, 127.5, 129.9, 134.2, 143.9. ESI-MS m/z = 317. m.p 130-132 oC.

1-aza-4,7-dithiacyclononane (III-6). Phenol (7.08 g, 0.075 mol) was dissolved in HBr in CH3COOH (33%, 90 mL) and compound III-5 (3.81 g, 0.012 mol) was added. The

solution was refluxed at 80 oC for 48 h and cooled to RT. Toluene (150 mL) was added to the dark solution and the solvent removed in vacuo. The black residue was added to

100 mL of water and washed repeatedly with CH2Cl2 until the organic layer was colorless. The combined organic layers were washed with 100 mL of water and the two aqueous portions were combined. NaOH was added to until pH~12 and the product was extracted with CHCl3 (5 x 100 mL). The combined organic layers were dried over

Na2SO4 and the solvent removed in vacuo yielding a tan sticky solid (1.90 g, 95%). Rf =

1 0.48 [silica gel: CH2Cl2]. H NMR (300 MHz, d6-DMSO) δ 3.28 (s, 1H), 3.03 (t, 4H),

13 2.89 (t, 4H), 2.70 (t, 4H). C NMR (75 MHz, d6-DMSO) δ 33.1, 33.2, 48.1. ESI-MS m/z = 163. m.p. 76-78 oC.

1-aza-4,7-dithiacyclononane rhodium trichloride (III-7). Compound III-6 (0.10 g,

0.0006 mol) was dissolved in 5 mL of EtOH and added to a solution of rhodium trichloride (0.13 g, 0.0006 mol) in EtOH. The solution was refluxed for 2 h yielding a brown precipitate. The solution was cooled to RT, filtered, and the brown solid was washed with EtOH and dried with Et2O yielding a dark tan solid (0.20 g, 88%).

Crystals suitable for X-ray diffraction were grown from a concentrated saline solution

1 by slow evaporation. H NMR (400 MHz, D2O) δ 3.83 (m, 3H), 3.54 (m, 3H), 3.31 (m,

13 3H), 3.10 (m, 3H), 2.99 (s, 1H). C NMR (75 MHz, D2O) δ 36.0, 36.5, 52.1. Anal.

Calcd. for C6H13N1S2Rh1Cl3 Theoretical: C 19.35, H 3.52, N 3.76. Found: C 18.97, H

3.63, N 3.54. decomposition point 265 oC. ESI-MS m/z = 373.

1-aza-4,7-dithiacyclononane Copper dichloride (III-8). Compound III-6 (0.064 g,

0.0004 mol) was added to 5 mL of EtOH and stirred into solution followed by addition

to a solution of CuCl2 (0.070 g, 0.0005 mol) in 5 mL of EtOH. An immediate green precipitate formed and the solution was stirred at RT overnight. The green precipitate was filtered and washed with 75 mL of EtOH (0.071 g, 61%). Crystals suitable for X- ray diffraction were grown from a concentrated CH3CN solution by slow evaporation.

Anal. Calcd. for C6H13N1S2Cu1Cl2 Theoretical: C 24.2, H 4.4, N 4.7. Found: C 23.45, H

4.36, N 4.4. decomposition point 147-150oC. ESI-MS m/z (-Cl) = 261.

Cell Lines. The human cancer cell lines OVCAR-3, MB157, and A549 were purchased from ATCC (Manassas, Virginia). The OVCAR-3 cell line was grown in RPMI 1640 media with 2 mM L-glutamine and modified to contain 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/L glucose, and 1.5 g/L sodium bicarbonate and was supplemented with

o 37 C with 5% CO2 and passed every 2-3 days. The A549 cell line was grown in F-12K media supplemented with 10% fetal bovine serum. The cells were grown at 37 oC with

5% CO2 and passed every 2-3 days.

MTT Assay. The MTT assay was purchased from Molecular Probes. The respective cell lines were grown to confluency and plated in 96-well plates at 5,000 cells per well in triplicate and allowed to incubate overnight. Compounds III-7 and III-8 were dissolved in DMSO to a concentration of 0.1 M and diluted into cell culture media to the desired testing concentrations. Media in each well was removed and replaced with

the fresh media containing test compounds. The test compounds were allowed to incubate for 24 hours after which the MTT protocol was followed. A stock solution of

MTT was prepared by adding 1 mL of PBS to the pre-weighed vial containing MTT. A

10 µL aliquot of this stock solution was added to each well and allowed to incubate for

4 hours. A stock solution of sodium dodecyl sulfate was prepared by adding 10 mL of

0.01 M HCl to the pre-weighed SDS vial. Following the 4 hour incubation period 100

µL of the SDS:HCl solution was added and incubated overnight. The optical density was read at 570 nm on Molecular Devices Spectramax M2 plate reader.

CHAPTER IV

SYNTHESIS AND CHARACTERIZATION OF SOME Ag(I) N-HETEROCYCLIC

CARBENES

4.1 Introduction

Because silver has been shown to be effective against various types of cancer cell lines in vitro,15,16,17,18,19,20,21,22 it only seems relevant to pursue the synthesis of a variety of silver complexes that will be stable enough to use systemically. The use of silver, systemically, will require a complex that is stable to proteins and salts in the bloodstream along with having the solubility necessary to remain in the blood long enough to be delivered to the cancer site.

The Youngs group has a long standing of synthesizing Ag(I) complexes of N- heterocyclic carbenes and we have showed that these complexes are formed quite readily and can be stable to simulated physiological conditions.101,102,103,104,105,106 Herein is reported the synthesis and characterization of a variety of imidazolium salts (N- heterocyclic carbene precursors) and their respective silver complexes, some of which may have future use as anticancer agents.

4.2 Synthesis of an Imidazolium Salt of Guanine

The biologically relevant nucleobase guanine, shown in Figure 4-1, was used as an imidazolium salt precursor for a couple of reasons. First of all, with a goal of the

Youngs group being to synthesize drugs for potential systemic use, in vivo toxicity is an area of importance. Since guanine is naturally occurring in DNA, our thought was that minor modifications of this compound would render it relatively non-toxic in the body.

Secondly, ongoing research in the group on silver complexes of caffeine had shown increased stability over previously reported complexes.101 Since guanine is structurally similar to caffeine, also shown in Figure 4-1, the thought was that silver complexes of guanine would also show added stability. Herein is reported the attempted synthesis of a silver acetate complex of guanine.

The imidazolium salt IV-1 was synthesized by adding guanine to 50 mL of dimethyl acetamide (DMA) and stirring for 30 minutes at 100 oC followed by the addition of 2.5 equivalents of dimethyl sulfate. The temperature was raised to 140 oC and the solution was stirred for 2 hours. Following cooling, 100 mL of MeOH was added along with NH4OH to a pH of approximately 8 resulting in a thick white precipitate. The precipitate was filtered, washed with hot MeOH, and dried in an oven overnight. The white solid was resolublized in 10 mL of aqueous NaOH solution and

HBr was added to pH 3. A yellow solid was precipitated by the addition of THF and filtered off to give IV-1 (eq. 4-1).

O O 1 1 H 5 5 N 6 N HN 6 N 7 7 2 8 8 3 2 3 O N 4 N H N N 4 N 9 2 9

Figure 4-1. Molecular Structure of Guanine (left) and Caffeine (right).

O O 1) CH3SO4 H 2) MeOH, NH OH N 4 N Br HN 3) NaOH, HBr, THF HN (4-1) DMA N H N N N H2N N 100 oC 2 3 h Equation 2-1. Synthesis of IV-1.

The imidazolium salt of guanine was characterized by NMR, mass spectrometry, elemental analysis, and X-ray crystallography. In the 1H NMR spectrum of IV-1, shown in Figure 4-3, there is a singlet at 3.68 ppm and 3.98 ppm for the methyl group protons off of the N7 and N9 positions, a singlet at 7.19 ppm for the NH2 protons, a singlet at 9.16 ppm for the imidazolium proton at the C8 position, and a singlet at 11.63 ppm for the proton at the N1 position. The 13C NMR spectrum, Figure 4-4, shows peaks at 31.7, 35.8, 139.3, 150.5, and 155.5 ppm, most notable of which is the peak at 139.3 representing the imidazolium C8 carbon. In the ESI-MS there is a peak that appears at m/z of 180 which represents the cationic portion of the imidazolium salt. Crystals suitable for X-ray diffraction were grown from a concentrated MeOH solution by slow evaporation and the solid state structure is shown in Figure 4-2.

100

Figure 4-2. X-ray crystal structure of IV-1. The thermal ellipsoids are shown at 50% probability.

101

-DMSO 6 in d IV-1 H NMR spectrum of 1

Figure 4-3.

102

-DMSO 6 d IV-1 C NMR spectrum of 13

Figure 4-4.

103

Various attempts were made to synthesize the silver complex of 7,9-

Dimethylguanine by using silver acetate and silver oxide. Characterization of the products from many reactions involving various reactions conditions was attempted.

Synthesis of the silver complex was never indicated by any form of characterization meaning that the slight deviation of structure from caffeine to guanine may have actually caused a decrease in stability.

4.3 Synthesis of an Imidazolium Salt of Guanosine

It was hypothesized that one of the reasons that the silver complex of guanine could not be synthesized was because during the silver reaction the N1 proton was being deprotonated. This idea came about because it is necessary to use a silver starting material that is basic to deprotonate the C8 proton in formation of the silver NHC complex. To combat this problem the silver complex of guanosine, shown in Figure 4-

5, was attempted. By forming the dimethylated version of guanosine, the N1 position would thus be protected by a methyl group.

The imidazolium salt of guanosine was synthesized similar to a published procedure.120 Briefly, guanosine was added to DMSO followed by the addition of three equivalents of K2CO3 and the solution was stirred for 10 minutes. Excess methyl iodide was added and the solution was stirred at room temperature overnight. The cloudy yellow solution was filtered and the DMSO was added to methylene chloride resulting in a white precipitate that was filtered yielding the white solid IV-2 (eq. 4-2).

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O 1 6 5 N HN 7 8 2 3 9 H N N 4 N 2 OH O OH HO

Figure 4-5. Molecular Structure of Guanosine

O O I HN N HN N K2CO3, CH3-I, CH2Cl2 (4-2) H N N N DMSO H N N N 2 OH RT 2 OH O O OH OH HO HO Equation 3-2. Synthesis of IV-2.

The imidazolium salt IV-2 was characterized by NMR, mass spectrometry, elemental analysis, and X-ray crystallography. All of the data obtained is consistent with the published data. The 1H spectrum, Figure 4-7, shows singlets at 2.53 ppm representing the methyl group protons at the N1 position, at 4.02 ppm for the methyl group protons at the N7 position, at 7.88 ppm for the NH2 protons, and at 9.37 ppm for the imidazolium proton at the C8 position. The protons of the sugar ring are represented by two multiplets at 3.61 and 3.68 ppm, two quartets at 4.15 and 4.37 ppm, and three doublets at 5.34, 5.64, and 5.83 ppm. In the 13C NMR spectrum there are peaks at 23.4, 28.2, 62.0, 70.5, 72.7, 85.8, 89.4, 149.7, 152.9, 154.9, and 175.8 ppm and

105

again the imidazolium C8 carbon is of note at 149.7 ppm. In the ESI-MS spectrum a peak arises at m/z of 312.3 representing the cationic portion of the molecule. X-ray suitable crystals were grown from a concentrated solution of MeOH/H20 by slow evaporation and the solid state structure is shown in Figure 4-6.

Figure 4-6. Crystal structure of IV-2. Thermal ellipsoids are shown at 50% probability.

106

-DMSO 6 d

in IV-2 H NMR spectrum of 1

Figure 4-7.

107

Again, various attempts at synthesizing a silver complex of 1,3-

Dimethylguanosine were unsuccessful. Since the N1 position was now protected and the silver complex was still unattained it was determined that guanine, or any of its derivatives, are not viable options for synthesizing systemically relevant silver complexes.

Though the silver complexes of these guanine derivatives were not synthesized, the imidazolium salt precursors have some comparable features. In the 1H NMR spectra the imidazolium proton resonances appear at 9.16 and 9.37 ppm for IV-1 and IV-2, respectively. For the 13C NMR spectra the imidazolium carbon resonances appear at

139.3 and 149.7, respectively and the shift is mainly due to the difference in anion.

4.4 Synthesis of 1-Carboxymethyl-3-methyl-4,5-dichloroimidazolium bromide

Due to the fact that we have shown increased silver NHC stability with 4,5- dichloro-1H-imidazole,105 the synthesis of a variety of derivatives is a necessary step in determining the most useful compound. One of these derivatives involves the addition of an acetic acid group to the imidazole ring in hopes of synthesizing a compound that is water soluble along with possessing increased complex stability.

1-Methyl-4,5-dichloroimidazole, shown in Figure 4-8, was added to 15 mL of water followed by the addition of 1-bromoacetic acid resulting in a clear solution that was refluxed for 3 days. The solution was then cooled to room temperature and the solvent removed in vacuo yielding a yellow oil. The oil was added to acetone resulting in the formation of a white solid, IV-3 (eq. 4-3).

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Cl N

N Cl

Figure 4-8. Molecular Structure of 1-methyl-4,5-dichloroimidazole

Cl Br O N Cl N Br OH Cl N (4-3) N H2O Cl 3 d O OH Equation 4-3. Synthesis of IV-3.

The imidazolium salt, IV-3, was characterized by NMR, mass spectrometry, elemental analysis, and X-ray crystallography. In the 1H NMR spectrum, shown in

Figure 4-10, singlets appear at 3.91 ppm for the methyl group protons, 5.08 ppm for the two methylene protons, and 9.06 ppm for the imidazolium proton. The 13C NMR spectrum, Figure 4-11, shows peaks at 35.1, 49.1, 119.9, 120.6, 136.5 representing the imidazolium carbon, and 169.0 ppm. In the ESI-MS a peak at m/z of 209 arises representing the cationic portion of the imidazolium salt. Crystals suitable for X-ray diffraction were grown from a concentrated solution of EtOH/H20 by slow evaporation.

The solid state structure of IV-3 is shown in Figure 4-9.

109

Figure 4-9. X-ray crystal structure of IV-3. Thermal ellipsoids are shown at 50% probability. Hydrogen atoms and anion are removed for clarity purposes.

110

O 2 in D IV-3 H NMRspectrum of 1

Figure 4-10.

111

O 2 in D IV-3 C NMR spectrum of 13

Figure 4-11.

112

The silver complex of IV-3 was attempted by reacting it with two and three equivalents of silver acetate in a variety of solvents. In all cases the product precipitated out of solution with the silver bromide and could not be recovered. The insolubility of the product can be attributed to the formation of oligomer type structures where the silver was bound through the carbene carbon and the acetate oxygen to form an extended network.

4.5 Synthesis of 1,3-dimethyl-4,5-diiodoimidazolium iodide

Because we have already shown that chlorides off of the four and five position of the imidazole ring enhance stability of the silver NHC complexes it seemed pertinent to explore what effects other halogens would have.105 The first derivative that was explored was the commercially available 1-Methyl-4,5-diiodo-1H-imidazole shown in

Figure 4-12.

1-Methyl-4,5-diiodoimidazole was added to CH3CN and heated resulting in a light yellow solution. Excess methyl iodide was added and the solution was refluxed overnight. A white precipitate formed, was filtered, and washed with CH3CN yielding

IV-4 (eq. 4-4).

I N

Figure 4-12. Molecular structure of 1-methyl-4,5-diiodoimidazole

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CH -I I I N 3 N + I (4-4) N CH3CN I N I

Equation 5-4. Synthesis of IV-4.

The characterization of IV-4 was carried out by NMR, mass spectrometry, elemental analysis, and X-ray crystallography. In the 1H NMR spectrum, shown in

Figure 4-14, two singlets arise, one at 3.82 ppm for the methyl group protons and one at

9.42 ppm for the imidazolium proton. Three different carbon peaks appear in the 13C

NMR spectrum, Figure 4-15, one at 38.7, 95.4, and 140.4 ppm which represents the imidazolium C8 carbon. The ESI-MS spectrum of IV-4 shows a peak at m/z of 348.9 which represents the cationic portion of the molecule. There is also a distinct iodine isotope pattern that appears in the ESI-MS which is to be expected for the iodide atoms on the 4 and 5 positions. Crystals suitable for X-ray diffraction were grown from a concentrated solution of EtOH/H20 by slow evaporation and the solid state structure of

IV-4 is shown in Figure 4-13.

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Figure 4-13. X-ray crystal structure of IV-4. Thermal ellipsoids are shown at 50% probability and hydrogen atoms are removed for clarity purposes.

115

-DMSO 6 in d IV-4 H NMR spectrum of 1

Figure 4-14.

116

-DMSO 6 in d IV-4 C NMR spectrum of 13

Figure 4-15.

117

4.6 Synthesis of 1,3-dimethyl-4,5-diiodoimidazole silver acetate

1,3-Dimethyl-4,5-diiodoimidazolium iodide was dissolved in a 1:1 mixture of

EtOH and H2O at room temperature. This solvent mixture was required because the imidazolium salt, IV-4, could only be solublized in this ratio of solvents. Two equivalents of silver acetate were then added resulting in an immediate yellow precipitate. The solution was stirred for 2 hours after which the precipitate was filtered off and the solvent removed in vacuo. The resulting white solid was washed in Et2O to remove the excess acetic acid yielding the white powdery solid IV-5, shown in Figure

4-16.

I N Ag O I N O IV-5

Figure 4-16. Molecular Structure of 1,3-dimethyl-4,5-diiodoimidazole silver acetate

The silver acetate structure IV-5 was characterized by NMR, mass spectrometry, and elemental analysis. The 1H NMR spectrum, Figure 4-18, shows singlets at 1.86 ppm for the methyl protons of the acetate group and 3.84 ppm for the N-methyl protons.

Indicative of silver complex formation is the loss of the peak at 9.42 ppm and the appearance of the peak at 1.86. In the 13C NMR spectrum, shown in Figure 4-19, peaks arise at 23.34, 39.13, 91.10, and 181.40 ppm. The most notable feature of this spectrum compared to its imidazolium salt is the loss of the peak at 140.42 and the appearance of the peak at 181.40 indicating that the Ag-C bond was formed. In the ESI-MS spectrum

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a peak arises at m/z of 454.8 which correlates to the silver complex with loss of the acetate moiety a common trend seen with these types of complexes. There is also a very distinct silver isotope pattern present which is indicative of the formation of the silver complex. The formation of X-ray quality crystals of IV-5 was attempted by concentrating a solution of the silver complex in in a variety of different solvents and allowing it to slowly evaporate. The only solvent that gave X-ray suitable crystals was water. However, the crystals that were isolated comprised the imidazolium cation with an acetate anion. From all of the data collected it appears that the silver complex may have been formed, but is unstable in H2O. The solid state structure of the crystals isolated is shown in Figure 4-17.

119

Figure 4-17. X-ray crystal structure of crystals isolated from IV-5 in H2O. Thermal ellipsoids are shown at 50% probability.

120

O 2 in D IV-5 H NMR spectrum of 1 Figure 4-18.

121

O 2 in D IV-5 C NMR spectrum of 13

Figure 4-19.

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4.7 Synthesis of 1,3,5-Tris(1,3-dimethyl-4,5-dichloroimidazolium iodide)-

2,4,6-trimethylbenzene

With the idea that silver NHC complexes possess anticancer activity it seemed interesting to explore the synthesis of complexes possessing more than one silver atom per molecule. This area of research was accomplished by using the starting material

1,3,5-Tris(bromomethyl)-2,4,6-trimethylbenzene, shown in Figure 4-20, which allows for the complexation of three silver atoms per molecule.

1-Methyl-4,5-dichloroimidazole was added to CH3CN followed by the addition of 1,3,5-Tris(bromomethyl)-2,4,6-trimethylbenzene. The solution was refluxed overnight resulting in a white precipitate. The solution was cooled to room temperature and the precipitate was filtered and washed with CH3CN yielding a white solid IV-6

(eq. 4-5).

Br Br

Figure 4-20. Molecular structure of 1,3,5-tris(bromomethyl)-2,4,6-trimethylbenzene

Cl Cl Br I Cl N N N I Cl N (4-5) Br Br N N CH3CN Reflux Cl N N Cl 24 h Cl I Cl

Equation 6-5. Synthesis of IV-6.

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The imidazolium salt IV-6 was characterized by NMR, mass spectrometry, elemental analysis, and X-ray crystallography. The 1H NMR spectrum, Figure 4-22, shows singlets at 2.30 ppm for the methyl group protons of the benzene ring, 3.89 ppm for the methyl group protons of the imidazole ring, 5.41 ppm for the protons of the methylene linker, and 9.66 ppm for the imidazolium protons. In the 13C NMR spectrum, shown in Figure 4-23, there are peaks at 16.1, 35.1, 47.8, 119.7, 121.2, 127.6,

134.1, and 143.3 ppm which represents the imidazolium carbon. The ESI-MS spectrum of IV-6 shows a peak at m/z of 874.7 which is representative of the sodiated form of the molecule. X-ray quality crystals were grown from a concentrated solution of EtOH by slow evaporation and the solid state structure is shown in Figure 4-21.

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Figure 4-21. X-ray crystal structure of IV-6. Thermal ellipsoids are shown at 50% probability. Hydrogen atoms and anions are removed for clarity purposes.

125

-DMSO 6 in d IV-6 H NMR spectrum of 1

Figure 4-22.

126

O 2 in D IV-6 C NMR spectrum of 13 Figure 4-23.

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4.8 Synthesis of 1,3,5-Tris(1,3-dimethyl-4,5-dichloroimidazole silver acetate)-

2,4,6-trimethylbenzene

The imidazolium salt IV-6 was added to MeOH resulting in a clear solution.

Six equivalents of silver acetate were added and an immediate yellow precipitate formed. The solution was allowed to stir for two hours at room temperature and the yellow precipitate was filtered. The solvent was removed in vacuo to give a clear viscous oil. The oil was stirred in Et2O overnight resulting in a white solid. The solid was filtered off and washed with Et2O yielding IV-7 (eq. 4-6).

O Cl N Cl Cl O Ag N I N N Cl I 2AgOAc O Cl (4-6) N N MeOH Cl Cl Cl 2 h O N N N N Ag N Cl I Cl Ag Cl N O O Cl

Equation 7-6. Synthesis of IV-7.

The silver acetate complex IV-7 was characterized by NMR, mass spectrometry, elemental analysis, and X-ray crystallography. The 1H NMR spectrum, Figure 4-25, shows singlets at 1.73 ppm for the methyl protons of the acetate group, 2.34 ppm for the methyl group protons of the benzene ring, 3.74 ppm for the methyl group protons of the imidazole ring, and 5.36 ppm for the protons of the methylene linker. An important indication of silver complex formation is the loss of the peak around 9 ppm where the imidazolium proton usually appears. In the 13C NMR spectrum, shown in Figure 4-26,

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there are peaks at 16.8, 23.4, 38.8, 47.1, 117.0, 117.5, 130.7, 139.0, 173.8, and 179.3 ppm. The loss of a peak around 143 and the appearance of the peak at 179.3 are indicative of silver complex formation. In the ESI-MS spectrum there is a peak at m/z of 716.9 which correlates to the silver complex with a loss of two of the silver atoms and three of the acetates. Indicative of silver complex formation is the silver isotope pattern that is also found in this spectrum. Crystals suitable for X-ray diffraction were grown from a concentrated EtOH solution by slow evaporation and the solid state structure is shown in Figure 4-24.

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Figure 4-24. X-ray crystal structure of IV-7. Thermal ellipsoids are shown at 50% probability and the hydrogen atoms are removed for clarity purposes.

130

-DMSO 6 in d IV-7 H NMR spectrum of 1

Figure 4-25.

131

-DMSO 6 in d IV-7 C NMR spectrum of 13

Figure 4-26.

132

In the solid state structure of IV-7 there are some interesting characteristics present. The average C(carbene)-Ag bond distance is approximately 2.08(1) Å, well within the expected range. Also, the average O(acetate)-Ag bond distance is 2.14(1) Å, also well within the expected range. However, there is a good degree of deviation from linearity in the C(carbene)-Ag-O(acetate) bond angles. The angle for Ag(1) is

165.10(18) Å, for Ag(2) is 158.20(18) Å, and for Ag(3) is 173.10(17) Å. Of the three the angle at Ag(2) is the least linear. This may be due to the fact that between Ag(2) and Ag(3) there is an Ag-Ag contact of 2.98(1) Å, a distance that is slightly too long to be an Ag-Ag bond, but well within the van der waals radius of 3.4 Å. Since this Ag-Ag interaction is seen in the asymmetric unit of the solid state it was necessary to understand whether this was caused by packing forces. If so, it would be expected that a packing diagram of the unit cell would show that Ag(3) also has a short contact to another molecule in the cell. The packing diagram of the cell is shown in Figure 4-27.

From this figure it is obvious that there is no Ag-Ag contact involving Ag(3).

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Figure 4-27. Packing diagram of the solid state structure of IV-7.

4.9 Conclusion

A variety of imidazolium salts, two from the naturally occurring guanine and three from the halogenated imidazoles, have been synthesized leading to a new tripodal silver acetate system that may have future use as an anticancer agent. This new silver acetate complex possesses three silver atoms per molecule which may lead to better activity than the previously reported silver complexes.

The salts that were synthesized from guanine, and its derivative guanosine, were explored due to their similarity to caffeine. It was believed that since silver complexes

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of caffeine had showed increased stability, and guanine is naturally occurring, this would be an excellent candidate for in vivo use. Contrary to this belief the silver complexes could not be synthesized due to their lack of stability.

The imidazolium salts synthesized from the halogenated imidazoles were explored because we have shown a halogenated silver complex to be the most stable to date. Since 4,5-dichloro-1H-imidazole has shown to be the most stable, the acetic acid derivative was explored as a compound with increased hydrophilicity, however its silver complex could not be obtained. 4,5-diiodo-1H-imidazole was explored to determine what effect the change in halogen had on the stability. Since the silver complex of the diiodo system was not stable in water it was determined to be less stable than its dichloro derivative. The tripodal compound was synthesized to form a compound that could possess more than one silver atom per molecule. In using the tri-brominated benzene derivative a tripodal silver acetate complex involving 4,5-dichloro-1H- imidazole was synthesized and may have use as a future anticancer agent.

4.10 Experimental

General. The starting materials 1-bromoacetic acid, methyl iodide, silver acetate, guanine, and guanosine were purchased from Sigma-Aldrich. 4,5-dichloroimidazole and 1-methyl-4,5-diiodoimidazole were purchased from Combi-Blocks USA. 1,3,5-

Tris(bromomethyl)-2,4,6-trimethylbenzene was purchased from Alfa Aesar. All chemicals were used without further purification. 1H NMR data was obtained using either a Varian 300 or 400 MHz instrument. 13C NMR data was obtained using a

135

Varian 300 MHz instrument. The spectra were referenced to the residual protons and the 13C signals of the deuterated solvents. Mass spectrometry data was collected from either the mass spectrometry laboratory at the University of Akron or at the Mass

Spectrometry and Proteomics Laboratory at The Ohio State University. Elemental analysis data was obtained from the University of Illinois, Urbana-Champaign. Melting points were taken on a Thomas Scientific Capillary Melting Point Apparatus.

X-ray crystallography. Crystals of IV-1 through IV-7 were coated in paratone oil, mounted on a CryoLoop™ and placed on a goniometer under a stream of nitrogen. X- ray data were collected using a Bruker Apex CCD diffractometer with graphite- monochromated Mo Kα radiation (λ= 0.71073 Å). The data was integrated using

7, 9-Dimethyl-guaninium-bromide (IV-1).121 Guanine (5 g, 0.033 mol) was added to

50 mL of dimethylacetamide and stirred for 0.5 h at 100 oC followed by the addition of

o (CH3)2SO4 (10 g, 0.077 mol). The temperature was raised to 140 C and the solution was stirred for 2 h. The solution was cooled to RT and 100 mL of MeOH was added followed by the addition of concentrated NH4OH to pH 8 resulting in a thick white precipitate. The precipitate was filtered, washed with 50 mL of hot MeOH, and dried in the oven overnight. The white solid was resolublized in 10 mL of aqueous NaOH

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followed by the addition of concentrated HBr to pH 3 and the solution was added to 50 mL of THF yielding a yellow precipitate that was filtered (6.95 g, 81%). X-ray suitable crystals were grown from a concentrated MeOH solution by slow evaporation. 1H NMR

(300 MHz, d6-DMSO) δ 3.68 (s, 3H), 3.98 (s,3H), 7.19 (s,2H), 9.16 (s, 1H), 11.63 (s,

13 1H). C NMR (75 MHz, D2O) δ 31.70, 35.87, 139.28, 150.48, 155.54. ESI-MS m/z =

180. Anal. Calcd. for C7H10N5O1Br1 Theoretical: C 32.35, H 3.87, N 26.94. Found: C

31.38, H 3.67, N 26.40. M.P. 285 oC

1,7-Dimethyl-guanosinium-iodide (IV-2). Guanosine (5 g, 0.017 mol) was added to a solution of K2CO3 (6 g, 0.043 mol) in 50 mL of DMSO and stirred for 10 minutes.

Methyl iodide (2.5 mL, 0.040 mol) was added and the solution was stirred overnight.

The cloudy yellow solution was filtered through celite and the DMSO was added slowly to 400 mL of CH2Cl2 causing a white precipitate that was filtered (3.97 g, 55%). X-ray quality crystals were grown from a concentrated solution of MeOH/H2O by slow

1 evaporation. H NMR (300 MHz, d6-DMSO) δ 2.53 (s, 3H), 3.61 (m, 1H), 3.68 (m,

1H), 4.02 (s, 3H), 4.15 (q, 1H), 4.37 (q, 1H), 5.11 (t, 1H), 5.34 (d, 1H), 5.64 (d, 1H),

13 5.83 (d, 1H), 7.88 (s, 2H), 9.37 (s, 1H). C NMR (75 MHz, d6-DMSO) δ 23.42, 28.17,

62.05, 70.55, 72.73, 85.81, 89.41, 149.73, 152.91, 154.90, 175.77. ESI-MS m/z =

312.3. Anal. Calcd. for C12H18N5O5I1 Theoretical: C 32.81, H 4.13, N 15.94. Found: C

31.82, H 3.92, N 15.62.

1-Carboxy methyl-3-methyl-4,5-dichloroimidazolium bromide (IV-3). 1-methyl-

4,5-dichloroimidazole (0.21 g, 0.0014 mol) was added to 15 mL of water followed by the addition of bromoacetic acid (0.21 g, .0015 mol) resulting in a clear solution which

137

was refluxed for 3 d. The solution was cooled to RT and the solvent removed in vacuo yielding a yellow oil. The oil was added to acetone resulting in the formation of a white solid (0.20 g, 50%). X-ray quality crystals were grown from a concentrated solution of

1 H2O by slow evaporation. H NMR (300 MHz, D2O) δ 3.91 (s, 3H), 5.08 (s, 2H), 9.06

13 (s, 1H). C NMR (75 MHz, D2O) 35.08, 49.12, 119.94, 120.62, 136.46, 169.01. ESI-

MS m/z = 209. Anal. Calcd. for C6H7N2O2Cl2Br Theoretical: C 24.85, H 2.40, N 9.60.

Found: C 24.83, H 2.41, N 9.37. M.P. 210 oC.

1,3-Dimethyl-4,5-diiodo-imidazolium iodide (IV-4). 1-methyl-4,5-diiodo-1H- imidazole (4.9 g, 0.0147 mmol) was added to 200 mL of CH3CN and heated resulting in a light yellow solution. Iodomethane (5 mL, 0.0735 mmol) was added and the solution was brought to reflux and allowed to stir overnight. The white precipitate that formed was filtered and washed with CH3CN yielding a white solid (6.6 g, 94%). X-ray quality crystals were grown from a concentrated solution of EtOH/H2O by slow evaporation.

1 13 H NMR (300 MHz, d6-DMSO) δ 3.82 (s, 6H), 9.42 (s, 1H). C NMR (75 MHz, d6-

DMSO) δ 38.76, 95.36, 140.42. ESI-MS m/z = 348.9. Anal. Calcd. for C5H7N2I3

Theoretical: C 12.62, H 1.48, N 5.88. Found: C 12.61, H 1.24, N 5.68. M.P. 254-256 oC.

1,3-Dimethyl-4,5-diiodo-imidazole silver acetate(IV-5). IV-4 (0.95 g, 0.002 mol) was dissolved in a 1:1 mixture of EtOH/H2O at room temperature. AgOAc (0.67 g, 0.004 mol) was added causing an immediate precipitate. The solution was stirred for 2 hours and the precipitate was filtered off. The solvent was removed in vacuo and the white

1 solid was washed with Et 2O yielding a white solid (0.77 g, 75%). H NMR (300 MHz,

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13 D2O) δ 1.86 (s, 3H), 3.84 (s, 6H). C NMR (75 MHz, D2O) δ 23.34, 39.13 91.10,

181.40. ESI-MS m/z = 454.8. Anal. Calcd. for C7H9N2O2I2Ag1 Theoretical: C 16.33, H

1.76, N 5.44. Found: C 18.19, H 2.12, N 5.41. M.P. 153-155 oC.

1,3,5-Tris(1,3-dimethyl-4,5-dichloroimidazolium iodide)-2,4,6-trimethylbenzene

(IV-6). 1-methyl-4,5-dichloroimidazole (0.45 g, 0.003 mol) was added to 40 mL of

CH3CN followed by the addition of 1,3,5-Tris(bromomethyl)-2,4,6-trimethylbenzene

(0.40 g, 0.001 mol). The solution was allowed to reflux overnight resulting in a white precipitate. The solution was cooled to RT and the precipitate was filtered and washed with CH3CN yielding a white solid (0.25 g, 30%). X-ray quality crystals were grown

1 from a concentrated solution of EtOH by slow evaporation. H NMR (300 MHz, d6-

DMSO) δ 2.30 (s, 9H), 3.89 (s, 9H), 5.41 (s, 6H), 9.66 (s, 3H). 13C NMR (75 MHz,

D2O) δ 16.09, 35.07, 47.83, 119.67, 121.23, 127.58, 134.08, 143.31. ESI-MS m/z (+

Na) = 874.7. Anal. Calcd. for C24H27N6Cl6Br3 Theoretical: C 33.83, H 3.19, N 9.86.

Found: C 32.90, H 3.26, N 9.74. M.P. 228-229 oC..

1,3,5-Tris(1,3-dimethyl-4,5-dichloroimidazole silver acetate)-2,4,6- trimethylbenzene (IV-7). IV-6 (0.10 g, 0.00012 mol) was added to MeOH resulting in a clear solution. AgOAc (0.12 g, 0.0007 mol) was added and an immediate yellow precipitate formed. The solution was stirred at RT for 2 hours and the precipitate was filtered. The solvent was removed in vacuo to give a clear viscous oil. The oil was stirred in Et2O overnight resulting in a white solid. The solid was filtered and washed with Et2O yielding a white solid (0.08 g, 60%). X-ray quality crystals were grown from

1 a concentrated solution of EtOH by slow evaporation. H NMR (300 MHz, d6-DMSO)

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13 δ 1.73 (s, 9H), 2.34 (s, 9H), 3.74 (s, 9H), 5.36 (s, 6H). C NMR (75 MHz, d6-DMSO)

δ 16.79, 23.37, 38.85, 47.12, 117.02, 117.54, 130.68, 139.03, 173.82, 179.34. ESI-MS m/z = 716.9. Anal. Calcd. for C30H33N6Cl6O6Ag3 Theoretical: C 32.37, H 2.18, N 7.55.

Found: C 32.85, H 3.17, N 7.68. M.P. 118 oC.

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CHAPTER V

CONCLUSION

With the emergence of cisplatin and carboplatin, the presence of metal-based anticancer drugs in the clinical setting is becoming increasingly important for a variety of reasons. A major reason is that the platinum-based drugs currently used are extremely toxic to most patients and usually lead to discontinuation of therapy. Another major reason is that, though these platinum drugs are active, resistance often occurs leading to relapse of the tumors. Lastly, each and every day cancer is becoming a bigger endemic disease throughout the world and new effective drugs are a necessity.

This study focused on the exploration of new non-platinum anticancer drugs for use against a variety of cancers. In chapter two the research focused on Ag(I) N- heterocyclic carbene complexes for the treatment of ovarian, breast, cervical, melanoma, renal , and colon cancer. In chapter three the focus was on Rh(III) and

Cu(II) and their complexation to a thiaether ligand for the treatment of ovarian, breast, and lung cancer. Lastly, chapter four is focused on the synthesis and characterization of some new silver drugs that may have potential future use as anticancer drugs.

The Ag(I) N-heterocyclic carbene complexes II-4, II-5, and II-6 focused on in chapter two showed impressive anticancer activity against all cancers tested except cervical in vitro. In a preliminary in vivo study metal complex II-4 appears to be active

141

against ovarian cancer when injected at the tumor site. These preliminary results are important because they indicate that if these drugs reach the tumor site they will have a major effect. These silver complexes were chosen for this study because one of the major goals of this research project was to develop a systemically viable drug, and to date silver complexes of this type are the most stable. For these reasons we believe that

Ag(I) N-heterocyclic carbene complexes may be a future replacement to the platinum- based drugs in the clinical setting.

The Rh(III) and Cu(II) work that was focused on in chapter three showed some mixed results. As previously stated, both of these metals possess anticancer activity against certain cancers. However, from the work done in this study only the copper complex III-8 was active enough against the ovarian, breast, and lung cancers to pursue in an in vivo model. The rhodium complex III-7 was not very active even at higher concentrations. It is thought that the reason for the low activity of the rhodium complex was the lack of solubility and the inherently high stability of the complex. Though the copper complex had comparable activity in vitro when compared to the silver complexes from chapter two, preliminary in vivo toxicity studies of III-8 showed it to be extremely toxic even at low doses. Due to this data metal complexes of Rh(III) and

Cu(II) were no longer pursued as potential drugs in this project.

In chapter four a variety of imidazolium salts were synthesized in hopes of developing a library of silver complexes to test their stability and cancer activity.

Unfortunately, only one silver complex could unambiguously be characterized. Silver complex IV-7 may be an improved compound, though, because it possesses the ability

142

to deliver three silver atoms per molecule. Theoretically this would make it three times more efficient than the silver complexes in chapter two, however, to date these results have not been determined.

From this project it appears that silver N-heterocyclic carbene complexes are possible anticancer drugs for the treatment of certain types of cancer. Further studies need to be explored to determine to what extent they can be utilized. Studies on the mechanism of action of these drugs are a necessity to determine if they kill cancer cells similarly to other metal complexes, or if a new pathway exists. Another major study that needs to be undertaken is to determine the in vivo stability of these drugs to understand if they can be injected systemically. If they are not stable enough for systemic delivery, then they could be encapsulated in nanoparticles to protect them from physiological conditions. Lastly, an in vivo toxicity study would be an important piece of data to determine clinically relevant doses and what parts of the body will be most affected. In all, it seems relevant to continue to pursue these types of drugs to help combat the growing cancer problem.

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BIBLIOGRAPHY

1 Rosenberg, B.; Van Camp, L.; Krigas, T. Nature, 1965, 205, 698.

2 Lebwohl, D.; Canetta, R. Euro. J. of Cancer. 1998, 34, 1522.

3 Wang, K.; Lu, J.; Li, R. Coord. Chem. Rev. 1996, 151, 53.

4 Johnson, D. Chest. 1999, 116, S525.

5 McGuire, W.; Ozols, R. Semin. Oncol. 1998, 25, 340.

6 Huisman, C.; Postmus, P.; Giaccone, G. Cancer Treat. Rev. 1999, 25, 199.

7 Thigpen, T. Am. Soc. Clin. Oncol. 1999.

8 Reedijk, J. Chem. Comm. 1996, 801.

9 Abu-Surrah, A. Mini-Rev. Med. Chem. 2007, 7, 203.

10 Lansdown, A. J. Wound Care. 2002, 11, 173.

11 Drake, P. Ann. Occup. Hyg. 2005, 49, 575.

12 Boswald, M.; Mende, K.; Bernschneider, W.; Bonakdar, S.; Ruder, H.; Kissler, H. Infection. 1999, 27, 38.

13 Brutel de la Riviere, A.; Dossche, K.; Binbaum, D.; Hacker, R. J. Heart Valve Disc. 2000, 9, 123.

14 Hostynyk, J.; Hinz, R.; Lorence, C.; Price, M.; Guy, R. Tox. 1993, 171.

15 Berners-Price, S.; Johnson, R.; Giovenella, A.; Faucette, L.; Mirabelli, C.; Sadler, P. J.Inorg. Biochem. 1988. 33, 285.

16 Liu, J.; Galetis, P.; Farr, A.; Maharaj, L.; Samarasinha, H.; McGechan, A.; Baguley, B.; Bowen, R.; Berners-Price, S.; McKeage, M. J. Inorg. Biochem. 2008, 102, 303.

144

17 Zhu, H.; Zhang, X.; Liu, X.; Wang, X.; Liu, G.; Usman, A.; Fun, H. Inorg. Chem. Comm. 2003, 6, 1113.

18 Liu, Z.; You, Z.; Zhu, H.; Tan, M. Inorg. Chem. Comm. 2004, 7, 1292.

19 Zachariadis, P.; Hadjikakou, S.; Hadjiliadis, N.; Skoulika, S.; Michaleides, A.; Balzarini, J.; de Clerq, E. Eur. J. Inorg. Chem. 2004, 1420.

20 Gottscaldt, M.; Pfeifer, A.; Koth, D.; Gorls, H.; Dahse, H.; Mollmann, U.; Obata, M.; Yano. S. Tet. 2006, 62, 11073.

21 Thati, B.; Noble, A.; Creaven, B.; Walsh, M.; McCann, M.; Kavanagh, K.; Devereux, M.; Egan, D. Cancer Lett. 2007, 248, 321.

22 Thati, B.; Noble, A.; Creaven, B.; Walsh, M.; McCann, M.; Kavanagh, K.; Devereux, M.; Egan, D. Cancer Lett. 2007, 250, 128.

23 Egan, D. Inorg. Chim. Acta. 2006, 359, 3976.

24 Hidalgo, E.; Domiguez, C. Tox. Lett. 1998, 98, 169.

25 Smith, J.; Martin, L. Proc. Natl. Acad. Sci 1973, 70, 1263.

26 Hughes, R.; Bear, L.; Kimball, A. Proc. Amer. Assoc. Cancer Res. 1972, 13, 120.

27 Pruchnik, F.; Dus, D. J. Inorg. Biochem. 1996, 61, 55.

28 De Souza, A.; Najjar, R.; Glikmanas, S.; Zyngier, S. J. Inorg. Biochem. 1996, 64, 1.

29 Pruchnik, F.; James, B.; Kvintovics, P. Can. J. Chem. 1986, 64, 936.

30 Rempel, G.; Legzdins, P.; Smith, H.; Wilkinson, G. Inorg. Synth. 1972, 13, 90.

31 Oyama, G. Proc. Soc. Exper. Biol. Med. 1956, 9, 305.

32 Erck, A.; Rainen, L.; Whileyman, J.; Bear, J. Proc. Soc. Exp. Biol. Med. 1974, 145, 1278.

33 Bear, J.; Gray, H.; Rainen, L.; Chang, I.; Howard, R.; Serio, G.; Kimball, A. Cancer Chemother. Rep. Part 1. 1975, 59, 611.

34 Rainen, L.; Howard, R.; Kimball, A.; Bear, J. Inorg. Chem. Trans. 1979, 14, 2752.

35 Chifotides, H.; Koshlap, K.; Perez, L.; Dunbar, K. J. Am.Chem. Soc. 2003, 125, 10703. 145

36 Chifotides, H.; Dunbar, K. Acc. Chem. Res. 2005, 38, 146.

37 Chifotides, H.; Koshlap, K.; Perez, L.; Dunbar, K. J. Am. Chem. Soc. 2003, 125, 10714.

38 Clarke, M.; Zhu, F.; Frasca, D. Chem. Rev. 1999, 99, 2511.

39 Jackson, B.; Barton, J. J. Am. Chem. Soc. 1997, 119, 12986.

40 Jackson, B.; Alekseyev, V.; Barton, J. Biochem. 1999, 38, 4655.

41 Odom, D.; Parker, C.; Barton, J. Biochem. 1999, 38, 5155.

42 Pruchnik, F.; Jakimowicz, P.; Ciunik, Z.; Zakrzewska-Czerwinska, J.; Opolski, A., Wietrzyk, J.; Wojdat, E. Inorg. Chim. Acta. 2002, 334, 59.

43 Clarke, M. Met. Ions Biol. Syst. 1980, 11, 231.

44 Keppler, B.; Henn, M.; Juhl, U.; Berger, M.; Niebl, R.; Balzer, W. Progr. Clin. Biochem. Med. 1989, 10, 41.

45 Keller, H.; Keppler, B. US Patent, 4843069, 1989.

46 Mestroni, G.; Alessio, E.; Sava, G. Int. Patent PCT C 07F 15/00, A61K 31/38, WO 98/00431, 1998.

47 Alessio, E.; Mestroni, G.; Bergamo, A.; Sava, G. Curr. Top. Med. Chem. 2004, 4, 1525.

48 Morris, R.; Aird, R.; Murdoch, P.; Chen, H.; Cummings, J.; Hughes, N.; Parsons, S.; Parkin, A.; Boyd, G.; Jodrell, D.; Sadler, P. J. Med. Chem. 2001, 44, 3616.

49 Hotze, A.; Bacac, M.; Velders, A.; Jansen, B.; Kooijman, H.; Spek, A.; Haasnoot, J.; Reedijk, J. J. Med. Chem. 2003, 46, 1743.

50 Vliet, P.; Toekimin, S.; Haasnoot, J.; Reedijk, J.; Novakova, O.; Vrana, O.; Brabec, V. Inorg. Chim. Acta. 1995, 231, 57.

51 Clarke, M.; Zhu, F.; Frasca, D. Chem. Rev. 1999, 99, 2511.

52 Kapitza, S.; Pongratz, M.; Jakupec, M.; Heffeter, P.; Berger, W.; Lackinger, L.; Keppler, B.; Marian, B. J. Cancer Res. 2005, 131, 101.

53 Kalinowski, D.; Richardson, D. Pharmacol. Rev. 2005, 57, 547.

146

54 Aouad, F.; Florence, A.; Zhang, Y.; Collins, F.; Henry, C. Inorg. Chim. Acta 2002, 339, 470.

55 Richardson, D. Curr. Med. Chem. 2005, 12, 2711.

56 Blatt, J.; Stitely, S. Cancer Res. 1987, 47, 1749.

57 Kalinowski, D.; Yu, Y.; Sharpe, P.; Islam, M.; Liao, Y.; Lovejoy, D.; Kumar, N.; Bernhardt, P.; Richardson, D. J. Med. Chem. 2007, 50, 3716.

58 Asayama, S.; Kasugai, N.; Kubota, S.; Nagaoka, S.; Kawakami, H. J. Inorg. Biochem. 2007, 101, 261.

59 Buss, J.; Greene, B.; Turner, J.; Torti, F.; Torti, S. Curr. Top. Med. Chem. 2004, 4, 1623.

60 Stevens, R.; Jones, D.; Micozzi, M.; Taylor, P. New Eng. J. Med. 1988, 319, 1047.

61 Petering, D. Metal Ions Biol. Med. 1980, 11, 197.

62 Gokhale, N.; Padhye, S.; Padhye, S.; Anson, C.; Powell, A. Inorg. Chim. Acta 2001, 319, 90.

63 Collins, M.; Ewing, D.; Mackenzie, G.; Sinn, E.; Sandbhor, U.; Padhye, S.; Padhye, S. Inorg. Chem. Comm. 2000, 3, 453.

64 Zou, G.; Cai, X.; Pan, N. Biometals. 2007, 20, 1.

65 Somasundaram, K. J. Med. Chem. 2005, 48, 977.

66 Butte, T. Immun. Today. 1994, 15, 7.

67 Levine, A. Cell. 1997, 88, 323.

68 Schibli, R.; Netter, R.; Waibel, R.; Abram, U.; Schbiger, A. Coord. Chem. Rev. 1999, 190-92, 901.

69 Garcia, R.; Paulo, A.; Domingos, A.; Santos, I.; Ortner, K.; Alberto, R. J. Am. Chem. Soc. 2000, 122, 11240.

70 Wei, L.; Banerjee, S.; Levadala, M.; Babich, J.; Zubieta, J. Inorg. Chem. Comm. 2003, 6, 1099.

71 Wang, W.; Spingler, B.; Alberto, R. Inorg. Chim. Acta 2003, 355, 386.

147

72 Bakir, M. Inorg. Chem. 2002, 332, 1.

73 Cabeza, N.; Garcia, A.; Moreno-Carretero, M.; Martos, J.; Exposito, R. J. Inorg. Biochem. 2005, 99, 1637.

74 Ma, D.; Che, C.; Siu, F.; Yang, M.; Wong, K. Inorg. Chem. 2007, 46, 740.

75 Baird, I. J. Med. Chem. 2006, 49, 5262.

76 Lim, I. J. Am. Chem. Soc. 2004, 126, 10271.

77 Maier, P. Cancer Chemother. Pharm. 1989, 23, 225.

78 Lummen, G.; Sperling, H.; Luboldt, H.; Otto, T.; Rubben, H. Cancer Chemother. Pharm. 1998, 42, 415.

79 Valadares, M.; Ramos, A.; Rehmann, F.; Sweeney, N.; Strohfeldt, M.; Quieroz, M. Euro. J. Pharm. 2006, 534, 264.

80 Pampillon, C.; Sweeney, N.; Strohfeldt, K.; Tacke, M. J. Organomet. Chem. 2007, 692, 2153.

81 Melendez, E. Crit. Rev. Onc. 2002, 42, 309.

82 Kopf-Maier, P. Naturwissenschaften. 1981, 68, 272.

83 Rivera, C. Met. Ions Bio. Med. 2000, 6, 580.

84 Harding, M. Curr. Med. Chem. 2000, 7, 1289.

85 Mirabelli, C.; Johnson, R.; Hill, D.; Faucette, L.; Girard, G. J. Med. Chem. 1986, 29, 218.

86 McKeage, M.; Maharaj, L.; Berners-Price, S. J. Coord. Chem.2002, 232, 127.

87 McKeage, M.; Berners-Price, S.; Galettis, P.; Brouwer, W.; Ding, L.; Zhaung, L.; Baguley, B. Cancer Chemother. Pharm. 2000, 46, 343.

88 Racham, O.; Nichols, S.; Leedman, S.; Berners-Price, S.; Filipovska, A. Biochem. Pharm. 2007, 74, 992.

89 Ortiz, A.; Dulk, H.; Brouwer, J.; Kooijman, H.; Spek, A.; Reedijk, J. J. Inorg. Biochem. 2007, 101, 1922.

90 Berners-Price, S.; Mirabelli, C.; Johnson, R. Cancer Res. 1986, 46, 486. 148

91 McKeage, M.; Berners-Price, S.; Maharaj, L. Coord. Chem. Rev. 2002, 232, 127.

92 Thati, B.; Noble, A.; Creaven, B.; Walsh, M.; McCann, M.; Kavanagh, K.; Devereux, M.; Egan, D. Cancer Letters. 2007, 248, 321-331.

93 Zhu, H.; Zhang, X.; Liu, X.; Wang, X.; Liu, G.; Usman, A.; Fun, H. Inorg. Chem. Comm. 2003, 6, 1113-1116.

94 Boswald, M.; Mende, K.; Bernschneider, W.; Bonakdar, S.; Ruder, H.; Kissler, H. Infection. 1999, 27, 38-42.

95 Brutel de la Riviere, A.; Dossche, K.; Birnbaum, D.; Hacker, R. J. Heart Valve Dis. 2000, 9, 123-129.

96 Karchmer, T.; Giannetta, E.; Muto, C.; Strain, B.; Farr, B. Arch. Intern. Med. 2000, 160, 3294-3298.

97 Tweden, K.; Cameron, J.; Razzouk, A.; Holmberg, W.; Kelly, S. J. Heart Valve Dis. 1997, 6, 553-561.

98 East, B.; Boddy, K.; Williams, E. Clin. Exp. Dematol. 1980, 5, 305-311.

99 Venugopal, B.; Luckey, T. Chemical Tox. Of Metals and Metalloids. 1978, 32-36.

100 Hostynyk, J.; Hinz, R.; Lorence, C.; Price, M.; Guy, R. Toxicology. 1993, 171-235.

101 Kascatan-Nebioglu, A.; Melaiye, A.; Hindi, K.; Durmus, S.; Panzner, M.; Hogue, L.; Mallett, R.; Hovis, C.; Coughenour, M.; Crosby, S.; Milsted, A.; Ely, D.; Tessier, C.; Cannon, C.; Youngs, W. J. Med. Chem. 2006, 49, 6811.

102 Garrison, J.; Youngs, W. Chem. Rev. 2005, 105(11), 3978-4008.

103 Garrison, J.; Simons, R.; Kofron, W.; Tessier, C.; Youngs, W. Chem. Comm. 2001, 18, 1780.

104 Garrison, J.; Simons, R.; Talley, J.; Wesdemiotis, C.; Tessier, C..; Youngs, W. Organometallics 2001, 20, 1276.

105 Hindi, K.; Siciliano, T.; Durmus, S.; Panzner, M.; Medvetz, D.; Hogue, L.; Hovis, C.; Hilliard, J.; Mallet, R.; Tessier, C.; Cannon, C.; Youngs, W. J. Med. Chem. 2008, 56, 1577.

106 Medvetz, D.; Hindi, K.; Panzner, M.; Ditto, A.; Yun, Y.; Youngs, W. Metal-Based Drugs. 2008, 1-7.

149

107 Lippard, S.; Sundquist, W.; Ahmed, K.; Hollis, S. Inorg. Chem. 1987, 26, 1524- 1528.

108 Wyllie, A.; Kerr, J.; Currie, A. Int. Rev. Cytology. 1980. 68, 251.

109 Jones, D.; McConkey, D.; Nicotera, P. j. Biol. Chem. 1989, 264, 6398.

110 Hechtfischer, A.; Marschall, M.; Helten, A.; Boswald, C.; Ewert, H. J. Gen. Vir. 1997, 78, 1327.

111 Houghton, P.; Fang, R. Nat. Prod. Res. 2007, 42, 377-87.

112 Sweeney, N.; Mendoza, O.; Mueller-Bunz, H.; Pampillon, C.; Rehmann, K.; Strohfeldt, K.; Tacke, M. J. Organomet. Chem. 2005, 690, 4537-4544.

113 Chifotides, H.; Koshlap, K.; Perez, L.; Dunbar, K. J. Am. Chem. Soc. 2003, 125, 10714-10724.

114 Briellman, M.; Kaderli, S.; Meyer, C.; Zuberbuhler, A. Helv. Cim. Acta. 1987, 70, 680-89.

115 Craig, A.; Kataky, R.; Matthews, R.; Parker, D.; Ferguson, G.; Lough, A.; Adams, H.; Bailey, N.; Schneider, N. J. Chem. Soc., Perkin Trans.2 1990, 9, 1523-31.

116 Evans, I.; Spencer, A.; Wilkinson, G. J. Chem. Soc. Dalton Tans. 1972, 204-8.

117 Blake, A.; Danks, J.; Fallis, I. Harrison, A.; Li, W.; Parsons, S.; Ross, S.; Whittaker, G.; Schroder, M. J. Chem. Soc., Dalton Trans. 1998, 3969.

118 Mattes, R.; Heinzel, U.; Henke, A. J.Chem. Soc., Dalton Trans. 1997, 4, 501-508.

119 Ren, T.; Liu, Y. Inorg. Chim. Acta. 2003, 348, 279-282.

120 Broom, A.; Townsend, L.; Jones, J.; Robins, R. Biochemistry. 1964, 3, 494-500.

121 Jones, J.; Robins, R. J. Am. Chem. Soc. 1962, 84, 1914-18.

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APPENDICES

151

APPENDIX A.

CRYSTAL STRUCTURE DATA FOR C6H12Cl3NRhS2 (III-7)

Table A-1. Crystal data and structure refinement for C6H12Cl3NRhS2 Empirical formula C6 H12 Cl3 N Rh S2 Formula weight 371.55 Temperature 100(2) K Wavelength 0.71073 Å Crystal system Orthorhombic Space group Pnma Unit cell dimensions a = 13.118(2) Å α= 90°. b = 11.896(2) Å β= 90°. c = 7.2330(13) Å γ = 90°. Volume 1128.7(3) Å3 Z 4 Density (calculated) 2.186 Mg/m3 Absorption coefficient 2.546 mm-1 F(000) 732 Crystal size 0.15 x 0.12 x 0.05 mm3 Theta range for data collection 3.11 to 28.31°. Index ranges -16<=h<=16, -15<=k<=15, -9<=l<=9 Reflections collected 9140 Independent reflections 1428 [R(int) = 0.0692] Completeness to theta = 28.31° 96.9 % Absorption correction Semi-empirical from equivalents Max. and min. transmission 0.8833 and 0.7013 Refinement method Full-matrix least-squares on F2 Data / restraints / parameters 1428 / 0 / 80 Goodness-of-fit on F2 1.182 Final R indices [I>2sigma(I)] R1 = 0.0507, wR2 = 0.1263 R indices (all data) R1 = 0.0543, wR2 = 0.1288 Largest diff. peak and hole 1.423 and -0.730 e.Å-3

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Table A-2. Atomic coordinates (x 104) and equivalent isotropic displacement parameters (Å2x 103) for III-7. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor. ______

x y z U(eq)

______

Rh(1) 8234(1) 7500 2605(1) 21(1) Cl(1) 6847(2) 7154(2) 511(3) 33(1) Cl(2A) 9260(5) 6048(7) 1099(10) 37(1) Cl(2B) 8954(6) 6115(7) 787(10) 39(1) S(1A) 7323(5) 6256(6) 4125(10) 37(1) S(1B) 7598(6) 6046(6) 4418(10) 33(1) N(1) 9357(4) 7500 4595(8) 32(1) C(1A) 9348(9) 6658(12) 5975(19) 43(3) C(2A) 8717(7) 5643(7) 5384(12) 60(4) C(3A) 6445(7) 7169(7) 5515(12) 35(3) C(1B) 9290(10) 6279(14) 5520(20) 47(3) C(2B) 8312(10) 6032(12) 6220(20) 47(3) C(3B) 6791(8) 6724(10) 5991(16) 34(3) ______

Table A-3. Bond lengths [Å] and angles [°] for III-7. ______

Rh(1)-N(1) 2.060(6) Rh(1)-S(1A) 2.197(7) Rh(1)-S(1A) 2.197(7) Rh(1)-Cl(2B) 2.310(8) Rh(1)-Cl(2B) 2.310(8) Rh(1)-S(1B) 2.326(7) Rh(1)-S(1B) 2.326(7) Rh(1)-Cl(1) 2.402(2) Rh(1)-Cl(1) 2.402(2) Rh(1)-Cl(2A) 2.445(7) Rh(1)-Cl(2A) 2.445(7) S(1A)-C(3A) 1.876(12) S(1A)-C(2A) 2.169(10) S(1B)-C(2B) 1.605(15) S(1B)-C(3B) 1.751(13) N(1)-C(1A) 1.414(13) N(1)-C(1A) 1.414(13) N(1)-C(1B) 1.602(16) 153

Table A-3. Bond lengths [Å] and angles [°] for III-7 (continued). ______

N(1)-C(1B) 1.602(16) C(1A)-C(2A) 1.526(16) C(1B)-C(2B) 1.409(19)

N(1)-Rh(1)-S(1A) 92.2(2) N(1)-Rh(1)-S(1A) 92.2(2) S(1A)-Rh(1)-S(1A) 84.7(4) N(1)-Rh(1)-Cl(2B) 96.0(2) S(1A)-Rh(1)-Cl(2B) 91.5(3) S(1A)-Rh(1)-Cl(2B) 171.04(18) N(1)-Rh(1)-Cl(2B) 96.0(2) S(1A)-Rh(1)-Cl(2B) 171.04(18) S(1A)-Rh(1)-Cl(2B) 91.5(3) Cl(2B)-Rh(1)-Cl(2B) 91.0(4) N(1)-Rh(1)-S(1B) 82.1(2) S(1A)-Rh(1)-S(1B) 91.38(18) S(1A)-Rh(1)-S(1B) 11.94(17) Cl(2B)-Rh(1)-S(1B) 176.6(2) Cl(2B)-Rh(1)-S(1B) 86.4(3) N(1)-Rh(1)-S(1B) 82.1(2) S(1A)-Rh(1)-S(1B) 11.94(17) S(1A)-Rh(1)-S(1B) 91.38(18) Cl(2B)-Rh(1)-S(1B) 86.4(3) Cl(2B)-Rh(1)-S(1B) 176.6(2) S(1B)-Rh(1)-S(1B) 96.1(4) N(1)-Rh(1)-Cl(1) 169.15(9) S(1A)-Rh(1)-Cl(1) 77.79(18) S(1A)-Rh(1)-Cl(1) 91.09(18) Cl(2B)-Rh(1)-Cl(1) 80.1(2) Cl(2B)-Rh(1)-Cl(1) 94.2(2) S(1B)-Rh(1)-Cl(1) 102.21(18) S(1B)-Rh(1)-Cl(1) 87.52(18) N(1)-Rh(1)-Cl(1) 169.15(9) S(1A)-Rh(1)-Cl(1) 91.09(18) S(1A)-Rh(1)-Cl(1) 77.79(18) Cl(2B)-Rh(1)-Cl(1) 94.2(2) Cl(2B)-Rh(1)-Cl(1) 80.1(2) S(1B)-Rh(1)-Cl(1) 87.52(18) S(1B)-Rh(1)-Cl(1) 102.21(18) Cl(1)-Rh(1)-Cl(1) 19.71(12) N(1)-Rh(1)-Cl(2A) 85.27(19) S(1A)-Rh(1)-Cl(2A) 176.3(3) S(1A)-Rh(1)-Cl(2A) 92.6(3) 154

Table A-3. Bond lengths [Å] and angles [°] for III-7 (continued). ______

Cl(2B)-Rh(1)-Cl(2A) 91.43(10) Cl(2B)-Rh(1)-Cl(2A) 10.79(19) S(1B)-Rh(1)-Cl(2A) 85.6(3) S(1B)-Rh(1)-Cl(2A) 166.86(17) Cl(1)-Rh(1)-Cl(2A) 104.89(16) Cl(1)-Rh(1)-Cl(2A) 90.87(16) N(1)-Rh(1)-Cl(2A) 85.27(19) S(1A)-Rh(1)-Cl(2A) 92.6(3) S(1A)-Rh(1)-Cl(2A) 176.3(3) Cl(2B)-Rh(1)-Cl(2A) 10.79(19) Cl(2B)-Rh(1)-Cl(2A) 91.43(10) S(1B)-Rh(1)-Cl(2A) 166.86(17) S(1B)-Rh(1)-Cl(2A) 85.6(3) Cl(1)-Rh(1)-Cl(2A) 90.87(16) Cl(1)-Rh(1)-Cl(2A) 104.89(16) Cl(2A)-Rh(1)-Cl(2A) 89.9(4) Cl(1)-Cl(1)-Rh(1) 80.15(6) C(3A)-S(1A)-C(2A) 119.1(5) C(3A)-S(1A)-Rh(1) 102.2(4) C(2A)-S(1A)-Rh(1) 88.8(3) C(2B)-S(1B)-C(3B) 80.2(7) C(2B)-S(1B)-Rh(1) 104.8(6) C(3B)-S(1B)-Rh(1) 103.9(5) C(1A)-N(1)-C(1A) 90.2(13) C(1A)-N(1)-C(1B) 110.3(11) C(1A)-N(1)-C(1B) 110.3(11) C(1B)-N(1)-C(1B) 130.2(13) C(1A)-N(1)-Rh(1) 119.2(6) C(1A)-N(1)-Rh(1) 119.2(6) C(1B)-N(1)-Rh(1) 104.6(6) C(1B)-N(1)-Rh(1) 104.6(6) N(1)-C(1A)-C(2A) 111.6(10) C(1A)-C(2A)-S(1A) 108.0(7) C(3A)-C(3A)-S(1A) 125.4(3) C(2B)-C(1B)-N(1) 112.9(11) C(1B)-C(2B)-S(1B) 103.8(11) S(1B)-C(3B)-C(3B) 117.4(4) ______

Symmetry transformations used to generate equivalent atoms:

#1 x,-y+3/2, z

155

Table A-4. Anisotropic displacement parameters (Å2x 103) for III-7. The anisotropic displacement factor exponent takes the form: -2π2[ h2 a*2U11 + ... + 2 h k a* b* U12 ]

______

U11 U22 U33 U23 U13 U12

______

Rh(1) 15(1) 33(1) 14(1) 0 1(1) 0 Cl(1) 20(1) 61(2) 18(1) -5(1) -4(1) 5(1) Cl(2A) 38(3) 43(2) 30(2) -19(2) 1(2) 15(2) Cl(2B) 53(4) 37(2) 26(2) -12(2) 6(2) -1(3) S(1A) 31(3) 48(3) 30(2) 8(2) -5(2) -23(2) S(1B) 31(3) 40(3) 27(2) 2(2) -3(2) -20(2) N(1) 19(3) 55(4) 21(3) 0 -1(2) 0 ______

Table A-5. Hydrogen coordinates (x 104) and isotropic displacement parameters (Å2x 10 3) for III-7. ______

x y z U(eq)

______

H(1A1) 9062 6974 7131 52 H(1A2) 10056 6415 6231 52 H(2A1) 8554 5171 6472 72 H(2A2) 9104 5182 4488 72 H(3A1) 5747 6938 5160 42 H(3A2) 6535 6938 6820 42 H(1B1) 9476 5706 4586 56 H(1B2) 9791 6232 6540 56 H(2B1) 8092 6608 7123 57 H(2B2) 8301 5285 6822 57 H(3B1) 6975 6468 7250 41 H(3B2) 6086 6468 5748 41 ______

156

APPENDIX B.

CRYSTAL STRUCTURE DATA FOR C6H13Cl2CuNS2 (III-8)

Table B-1. Crystal data and structure refinement for C6H13Cl2CuNS2. Empirical formula C6 H13 Cl2 Cu N S2 Formula weight 297.73 Temperature 100(2) K Wavelength 0.71073 Å Crystal system Monoclinic Space group P2(1)/n Unit cell dimensions a = 7.4990(7) Å α= 90°. b = 13.0417(11) Å β= 94.240(2)°. c = 10.6185(9) Å γ = 90°. Volume 1035.64(16) Å3 Z 4 Density (calculated) 1.910 Mg/m3 Absorption coefficient 2.973 mm-1 F(000) 604 Crystal size 0.13 x 0.05 x 0.03 mm3 Theta range for data collection 2.48 to 28.28°. Index ranges -9<=h<=9, -17<=k<=17, -14<=l<=13 Reflections collected 8854 Independent reflections 2423 [R(int) = 0.0293] Completeness to theta = 28.28° 94.5 % Absorption correction Semi-empirical from equivalents Max. and min. transmission 0.9161 and 0.7557 Refinement method Full-matrix least-squares on F2 Data / restraints / parameters 2423 / 0 / 109 Goodness-of-fit on F2 1.119 Final R indices [I>2sigma(I)] R1 = 0.0355, wR2 = 0.0777 R indices (all data) R1 = 0.0410, wR2 = 0.0800 Largest diff. peak and hole 1.172 and -0.396 e.Å-3

157

Table B-2. Atomic coordinates (x 104) and equivalent isotropic displacement parameters (Å2x 103) for III-8. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor. ______

x y z U(eq)

______

Cu(1) 931(1) 5107(1) 6670(1) 12(1) Cl(1) 2373(1) 3989(1) 5418(1) 16(1) Cl(2) -1197(1) 3973(1) 7211(1) 16(1) S(1) -619(1) 6441(1) 7587(1) 15(1) S(2) 3067(1) 5047(1) 8662(1) 14(1) N(1) 2566(3) 6249(2) 6043(2) 15(1) C(1) 300(4) 6384(2) 9214(3) 18(1) C(2) 2286(4) 6174(2) 9463(3) 17(1) C(3) 4805(4) 5563(2) 7743(3) 19(1) C(4) 4221(4) 6449(2) 6882(3) 17(1) C(5) 1508(4) 7213(2) 5811(3) 15(1) C(6) 552(4) 7524(2) 6967(3) 14(1) ______

Table B-3. Bond lengths [Å] and angles [°] for III-8. ______

Cu(1)-N(1) 2.070(3) Cu(1)-Cl(2) 2.2803(8) Cu(1)-Cl(1) 2.2960(8) Cu(1)-S(1) 2.3438(8) Cu(1)-S(2) 2.5587(8) S(1)-C(1) 1.813(3) S(1)-C(6) 1.814(3) S(2)-C(3) 1.815(3) S(2)-C(2) 1.816(3) N(1)-C(4) 1.496(4) N(1)-C(5) 1.497(4) C(1)-C(2) 1.518(4) C(3)-C(4) 1.517(4) C(5)-C(6) 1.521(4)

N(1)-Cu(1)-Cl(2) 171.81(7) N(1)-Cu(1)-Cl(1) 87.43(7) Cl(2)-Cu(1)-Cl(1) 95.89(3) N(1)-Cu(1)-S(1) 85.62(7) 158

Table B-3. Bond lengths [Å] and angles [°] for III-8 (continued). ______

Cl(2)-Cu(1)-S(1) 89.91(3) Cl(1)-Cu(1)-S(1) 168.79(3) N(1)-Cu(1)-S(2) 86.47(7) Cl(2)-Cu(1)-S(2) 100.31(3) Cl(1)-Cu(1)-S(2) 99.58(3) S(1)-Cu(1)-S(2) 88.77(3) C(1)-S(1)-C(6) 102.57(14) C(1)-S(1)-Cu(1) 101.55(10) C(6)-S(1)-Cu(1) 99.14(10) C(3)-S(2)-C(2) 102.76(15) C(3)-S(2)-Cu(1) 88.52(11) C(2)-S(2)-Cu(1) 99.11(10) C(4)-N(1)-C(5) 110.9(2) C(4)-N(1)-Cu(1) 114.76(18) C(5)-N(1)-Cu(1) 109.72(18) C(2)-C(1)-S(1) 118.2(2) C(1)-C(2)-S(2) 114.3(2) C(4)-C(3)-S(2) 114.8(2) N(1)-C(4)-C(3) 114.5(2) N(1)-C(5)-C(6) 111.6(2) C(5)-C(6)-S(1) 110.8(2) ______

Symmetry transformations used to generate equivalent atoms:

Table B-4. Anisotropic displacement parameters (Å2x 103) for III-8. The anisotropic displacement factor exponent takes the form: -2π2[ h2 a*2U11 + ... + 2 h k a* b* U12 ] ______

U11 U22 U33 U23 U13 U12

______

Cu(1) 15(1) 8(1) 12(1) -1(1) 1(1) -2(1) Cl(1) 20(1) 13(1) 15(1) -3(1) 2(1) 2(1) Cl(2) 19(1) 12(1) 18(1) 0(1) 4(1) -4(1) S(1) 13(1) 13(1) 20(1) 0(1) 2(1) 0(1) S(2) 14(1) 12(1) 16(1) 1(1) -1(1) 0(1) N(1) 17(1) 9(1) 18(1) 2(1) -5(1) 0(1) C(1) 24(2) 15(2) 17(2) 0(1) 6(1) 0(1) 159

Table B-4. Anisotropic displacement parameters (Å2x 103) for III-8. The anisotropic displacement factor exponent takes the form: -2π2[ h2 a*2U11 + ... + 2 h k a* b* U12 ] (continued) ______

C(2) 24(2) 15(2) 13(1) -2(1) 2(1) -1(1) C(3) 10(1) 20(2) 26(2) 4(1) 3(1) 1(1) C(4) 13(2) 15(2) 23(2) 3(1) 4(1) 0(1) C(5) 18(2) 12(1) 15(1) 1(1) 0(1) 0(1) C(6) 14(1) 10(1) 19(2) 3(1) 0(1) 0(1)

Table B-5. Hydrogen coordinates (x 104) and isotropic displacement parameters (Å2x 103) for III-8. ______

x y z U(eq)

______

H(1A) 38 7045 9619 22 H(1B) -355 5845 9647 22 H(2A) 2558 6088 10383 21 H(2B) 2959 6778 9194 21 H(3A) 5810 5796 8330 22 H(3B) 5258 5005 7221 22 H(4A) 4014 7057 7410 21 H(4B) 5208 6618 6348 21 H(5A) 616 7108 5088 18 H(5B) 2319 7773 5588 18 H(6A) -313 8079 6735 17 H(6B) 1434 7791 7626 17 ______

160

APPENDIX C.

CRYSTAL STRUCTURE DATA FOR C7H10BrN5O2 (IV-1)

Table C-1. Crystal data and structure refinement for C7H10BrN5O2. Empirical formula C7 H10 Br N5 O2 Formula weight 260.11 Temperature 100(2) K Wavelength 0.71073 Å Crystal system Triclinic Space group P-1 Unit cell dimensions a = 7.707(2) Å α= 67.532(4)°. b = 10.826(3) Å β= 78.589(5)°. c = 13.607(4) Å γ = 82.380(5)°. Volume 1026.4(5) Å3 Z 4 Density (calculated) 1.683 Mg/m3 Absorption coefficient 3.982 mm-1 F(000) 520 Crystal size 0.23 x 0.17 x 0.07 mm3 Theta range for data collection 1.64 to 28.38°. Index ranges -10<=h<=10, -14<=k<=14, -17<=l<=17 Reflections collected 8948 Independent reflections 4707 [R(int) = 0.0471] Completeness to theta = 28.38° 91.3 % Absorption correction Semi-empirical from equivalents Max. and min. transmission 0.7680 and 0.4333 Refinement method Full-matrix least-squares on F2 Data / restraints / parameters 4707 / 0 / 351 Goodness-of-fit on F2 1.028 Final R indices [I>2sigma(I)] R1 = 0.0460, wR2 = 0.1150 R indices (all data) R1 = 0.0579, wR2 = 0.1215 Largest diff. peak and hole 1.047 and -0.853 e.Å-3

161

Table C-2. Atomic coordinates ( x 104) and equivalent isotropic displacement parameters (Å2x 103) for IV-1. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor. ______

x y z U(eq)

______

Br(1) 7295(1) 874(1) 569(1) 18(1) Br(2) 8283(1) 9078(1) 4291(1) 18(1) O(1) -1242(4) 4220(3) 4075(2) 18(1) O(2) -222(3) 5789(3) 832(2) 18(1) O(3) 8090(4) 8379(3) 9592(2) 21(1) O(4) 7173(4) 1610(3) 5181(2) 23(1) N(1) 3157(4) 2100(3) 2677(2) 13(1) N(2) 491(4) 2787(3) 2299(2) 15(1) N(3) 2743(4) 5912(3) 774(3) 16(1) N(4) 4700(4) 4126(3) 1670(2) 14(1) N(5) 5693(5) 6216(4) 648(3) 19(1) N(6) 2023(4) 7952(3) 2432(2) 14(1) N(7) -578(4) 7223(3) 2624(2) 14(1) N(8) 1699(4) 4107(3) 4225(3) 15(1) N(9) 3590(4) 5927(3) 3450(2) 15(1) N(10) 4632(5) 3823(4) 4460(3) 19(1) C(1) 1489(5) 1753(4) 2835(3) 15(1) C(2) 3229(5) 3426(4) 1996(3) 12(1) C(3) 1561(5) 3862(4) 1760(3) 13(1) C(4) 1200(5) 5220(4) 1094(3) 15(1) C(5) 4388(5) 5385(4) 1047(3) 15(1) C(6) 4661(5) 1221(4) 3126(3) 14(1) C(7) -1384(5) 2788(4) 2255(3) 19(1) C(8) 380(5) 8283(4) 2182(3) 15(1) C(9) 2123(5) 6619(3) 3072(3) 12(1) C(10) 485(5) 6152(4) 3199(3) 14(1) C(11) 153(5) 4791(4) 3850(3) 14(1) C(12) 3305(5) 4649(4) 4030(3) 16(1) C(13) 3475(5) 8852(4) 2094(3) 17(1) C(14) -2450(5) 7206(4) 2580(3) 18(1)

162

Table C-3. Bond lengths [Å] and angles [°] for IV-1. ______

O(1)-C(11) 1.238(4) O(2)-C(4) 1.231(4) N(1)-C(1) 1.340(5) N(1)-C(2) 1.381(4) N(1)-C(6) 1.471(5) N(2)-C(1) 1.321(5) N(2)-C(3) 1.391(4) N(2)-C(7) 1.458(5) N(3)-C(5) 1.380(5) N(3)-C(4) 1.395(5) N(4)-C(5) 1.321(5) N(4)-C(2) 1.352(4) N(5)-C(5) 1.329(5) N(6)-C(8) 1.343(5) N(6)-C(9) 1.373(4) N(6)-C(13) 1.469(5) N(7)-C(8) 1.321(5) N(7)-C(10) 1.392(4) N(7)-C(14) 1.459(5) N(8)-C(12) 1.375(5) N(8)-C(11) 1.396(5) N(9)-C(12) 1.328(5) N(9)-C(9) 1.347(5) N(10)-C(12) 1.335(5) C(2)-C(3) 1.366(5) C(3)-C(4) 1.426(5) C(9)-C(10) 1.377(5) C(10)-C(11) 1.426(5)

C(1)-N(1)-C(2) 107.8(3) C(1)-N(1)-C(6) 126.4(3) C(2)-N(1)-C(6) 125.8(3) C(1)-N(2)-C(3) 107.8(3) C(1)-N(2)-C(7) 126.0(3) C(3)-N(2)-C(7) 126.2(3) C(5)-N(3)-C(4) 126.1(3) C(5)-N(4)-C(2) 112.1(3) C(8)-N(6)-C(9) 108.4(3) C(8)-N(6)-C(13) 126.4(3) C(9)-N(6)-C(13) 125.2(3) C(8)-N(7)-C(10) 107.7(3) C(8)-N(7)-C(14) 125.9(3) C(10)-N(7)-C(14) 126.2(3) 163

Table C-3. Bond lengths [Å] and angles [°] for IV-1 (continued). ______C(12)-N(8)-C(11) 126.0(3) C(12)-N(9)-C(9) 111.8(3) N(2)-C(1)-N(1) 110.1(3) N(4)-C(2)-C(3) 128.4(3) N(4)-C(2)-N(1) 124.5(3) C(3)-C(2)-N(1) 107.0(3) C(2)-C(3)-N(2) 107.2(3) C(2)-C(3)-C(4) 120.2(3) N(2)-C(3)-C(4) 132.5(3) O(2)-C(4)-N(3) 121.3(3) O(2)-C(4)-C(3) 128.8(3) N(3)-C(4)-C(3) 109.9(3) N(4)-C(5)-N(5) 120.1(3) N(4)-C(5)-N(3) 123.4(3) N(5)-C(5)-N(3) 116.5(3) N(7)-C(8)-N(6) 110.0(3) N(9)-C(9)-N(6) 125.1(3) N(9)-C(9)-C(10) 128.3(3) N(6)-C(9)-C(10) 106.6(3) C(9)-C(10)-N(7) 107.3(3) C(9)-C(10)-C(11) 120.2(3) N(7)-C(10)-C(11) 132.5(3) O(1)-C(11)-N(8) 121.5(3) O(1)-C(11)-C(10) 128.7(3) N(8)-C(11)-C(10) 109.9(3) N(9)-C(12)-N(10) 119.3(4) N(9)-C(12)-N(8) 123.8(3) N(10)-C(12)-N(8) 116.9(3) ______

Symmetry transformations used to generate equivalent atoms:

164

Table C-4. Anisotropic displacement parameters (Å2x 103) for IV-1. The anisotropic displacement factor exponent takes the form: -2π2[ h2 a*2U11 + ... + 2 h k a* b* U12 ]

______

U11 U22 U33 U23 U13 U12

______

Br(1) 22(1) 12(1) 15(1) -2(1) -3(1) 1(1) Br(2) 23(1) 12(1) 16(1) -2(1) -1(1) -1(1) O(1) 19(1) 14(1) 18(1) -2(1) -1(1) -7(1) O(2) 17(1) 16(1) 20(1) -4(1) -6(1) 3(1) O(3) 24(2) 14(1) 24(2) -4(1) -6(1) 0(1) O(4) 22(2) 17(1) 26(2) -4(1) -3(1) 0(1) N(1) 13(2) 10(1) 14(2) -3(1) -1(1) 0(1) N(2) 14(2) 16(2) 13(2) -4(1) 1(1) -1(1) N(3) 20(2) 10(2) 14(2) -2(1) -3(1) 1(1) N(4) 14(2) 15(2) 12(1) -4(1) 0(1) -4(1) N(5) 20(2) 15(2) 18(2) -2(1) -3(1) -2(1) N(6) 12(2) 15(2) 12(1) -3(1) -1(1) -3(1) N(7) 14(2) 14(2) 13(2) -3(1) -2(1) 0(1) N(8) 18(2) 11(2) 15(2) -3(1) -3(1) -2(1) N(9) 16(2) 13(2) 14(2) -2(1) -4(1) 1(1) N(10) 21(2) 14(2) 20(2) -2(1) -8(1) 3(1) C(1) 15(2) 14(2) 16(2) -6(2) -1(2) -1(1) C(2) 14(2) 13(2) 8(2) -4(1) 2(1) -2(1) C(3) 12(2) 14(2) 9(2) -1(1) -1(1) -2(1) C(4) 16(2) 16(2) 13(2) -6(2) 0(1) 1(1) C(5) 16(2) 15(2) 13(2) -6(2) -4(2) -1(1) C(6) 12(2) 15(2) 14(2) -4(2) -2(2) 3(1) C(7) 11(2) 19(2) 25(2) -6(2) -4(2) -3(2) C(8) 15(2) 17(2) 12(2) -4(2) -4(1) 2(2) C(9) 13(2) 12(2) 12(2) -5(1) 0(1) 1(1) C(10) 14(2) 14(2) 12(2) -5(1) -2(1) 1(1) C(11) 17(2) 13(2) 11(2) -5(1) -3(1) 1(1) C(12) 17(2) 17(2) 14(2) -7(2) -4(2) 2(2) C(13) 18(2) 15(2) 18(2) -3(2) -4(2) -6(2) C(14) 11(2) 20(2) 18(2) -1(2) -6(2) 1(2) ______

165

Table C-5. Hydrogen coordinates (x 104) and isotropic displacement parameters (Å2x 10 3) for IV-1. ______

x y z U(eq)

______

H(8) 2680(60) 6690(50) 480(40) 24(13) H(9) 5540(60) 7030(50) 190(40) 26(12) H(10) 6640(60) 5960(50) 820(40) 24(13) H(18) 1660(60) 3290(50) 4580(40) 22(12) H(19) 4490(70) 2980(50) 4780(40) 35(14) H(20) 5570(70) 4140(50) 4350(40) 32(14) H(1) 1020(60) 880(40) 3330(30) 23(12) H(2) 4990(60) 1600(40) 3610(30) 22(11) H(3) 4300(50) 330(40) 3590(30) 13(10) H(4) 5500(50) 1180(40) 2550(30) 13(10) H(5) -2110(60) 3420(40) 2620(30) 22(11) H(6) -1740(70) 1920(60) 2620(40) 41(15) H(7) -1460(70) 2940(50) 1530(40) 47(16) H(11) -80(50) 9120(40) 1770(30) 1(8) H(12) 4240(60) 8690(40) 1470(30) 20(11) H(13) 3060(50) 9800(40) 1740(30) 13(10) H(14) 4010(50) 8590(30) 2740(30) 2(8) H(15) -2670(60) 6550(40) 2350(30) 19(11) H(16) -2790(60) 8090(50) 2040(40) 26(12) H(17) -3190(60) 6990(40) 3300(30) 21(11) ______

166

APPENDIX D.

CRYSTAL STRUCTURE DATA FOR C12H18IN5O5 (IV-2)

Table D-1. Crystal data and structure refinement for C12H18IN5O5. Empirical formula C12 H18 I N5 O5 Formula weight 439.21 Temperature 100(2) K Wavelength 0.71073 Å Crystal system Orthorhombic Space group P2(1)2(1)2(1) Unit cell dimensions a = 7.1929(9) Å α= 90°. b = 12.5268(16) Å β= 90°. c = 17.130(2) Å γ = 90°. Volume 1543.5(3) Å3 Z 4 Density (calculated) 1.890 Mg/m3 Absorption coefficient 2.109 mm-1 F(000) 872 Crystal size 0.15 x 0.12 x 0.05 mm3 Theta range for data collection 2.01 to 28.29°. Index ranges -9<=h<=9, -16<=k<=16, -22<=l<=22 Reflections collected 13514 Independent reflections 3720 [R(int) = 0.0539] Completeness to theta = 28.29° 98.5 % Absorption correction Semi-empirical from equivalents Max. and min. transmission 0.9019 and 0.6493 Refinement method Full-matrix least-squares on F2 Data / restraints / parameters 3720 / 0 / 213 Goodness-of-fit on F2 1.252 Final R indices [I>2sigma(I)] R1 = 0.0420, wR2 = 0.0962 R indices (all data) R1 = 0.0422, wR2 = 0.0963 Absolute structure parameter 0.06(3) Largest diff. peak and hole 1.480 and -1.258 e.Å-3

167

Table D-2. Atomic coordinates (x 104) and equivalent isotropic displacement parameters (Å2x 103) for IV-2. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor. ______

x y z U(eq)

______

I(1) 4478(1) 9651(1) 438(1) 14(1) O(1) 6363(5) 6582(3) 1298(2) 13(1) O(2) 4618(6) 3826(3) -1826(2) 16(1) O(3) 1524(5) 6627(3) 1601(2) 15(1) O(4) 3988(6) 7338(3) 2657(2) 18(1) O(5) 8853(6) 5041(3) 1694(2) 19(1) N(1) 4193(6) 6018(4) -1028(2) 11(1) N(2) 4461(7) 6139(3) 242(2) 12(1) N(3) 5058(6) 3180(3) -589(2) 12(1) N(4) 4971(6) 4262(3) 559(2) 11(1) N(5) 5456(8) 2467(4) 653(2) 18(1) C(1) 4178(6) 6673(4) -429(3) 11(1) C(2) 4490(8) 4996(4) -735(3) 9(1) C(3) 4680(8) 5059(4) 56(3) 11(1) C(4) 4711(7) 4013(4) -1126(3) 12(1) C(5) 5147(7) 3329(4) 206(3) 12(1) C(6) 3771(8) 6292(4) -1838(3) 13(1) C(7) 4530(9) 6636(4) 1013(3) 12(1) C(8) 3249(7) 6110(4) 1611(3) 13(1) C(9) 4342(9) 6281(4) 2367(3) 14(1) C(10) 6358(8) 6213(5) 2103(3) 14(1) C(11) 7149(7) 5096(4) 2136(3) 14(1) C(12) 5278(9) 2099(4) -912(3) 20(1) ______

Table D-3. Bond lengths [Å] and angles [°] for IV-2. ______

O(1)-C(7) 1.407(7) O(1)-C(10) 1.454(6) O(2)-C(4) 1.224(6) O(3)-C(8) 1.400(6) O(4)-C(9) 1.437(6) O(5)-C(11) 1.442(7) N(1)-C(1) 1.315(7) N(1)-C(2) 1.391(6) 168

Table D-3. Bond lengths [Å] and angles [°] for IV-2 (continued). ______

N(1)-C(6) 1.461(6) N(2)-C(1) 1.345(6) N(2)-C(3) 1.399(7) N(2)-C(7) 1.460(6) N(3)-C(5) 1.377(6) N(3)-C(4) 1.414(6) N(3)-C(12) 1.471(6) N(4)-C(5) 1.322(7) N(4)-C(3) 1.335(7) N(5)-C(5) 1.342(7) C(2)-C(3) 1.365(6) C(2)-C(4) 1.411(7) C(7)-C(8) 1.527(7) C(8)-C(9) 1.530(7) C(9)-C(10) 1.521(8) C(10)-C(11) 1.512(7)

C(7)-O(1)-C(10) 110.0(4) C(1)-N(1)-C(2) 107.1(4) C(1)-N(1)-C(6) 126.4(4) C(2)-N(1)-C(6) 126.2(4) C(1)-N(2)-C(3) 107.7(4) C(1)-N(2)-C(7) 124.4(4) C(3)-N(2)-C(7) 127.9(4) C(5)-N(3)-C(4) 123.5(4) C(5)-N(3)-C(12) 119.5(4) C(4)-N(3)-C(12) 117.0(4) C(5)-N(4)-C(3) 112.5(4) N(1)-C(1)-N(2) 110.8(4) C(3)-C(2)-N(1) 108.7(4) C(3)-C(2)-C(4) 120.7(5) N(1)-C(2)-C(4) 130.5(4) N(4)-C(3)-C(2) 127.8(5) N(4)-C(3)-N(2) 126.5(4) C(2)-C(3)-N(2) 105.7(4) O(2)-C(4)-C(2) 128.8(5) O(2)-C(4)-N(3) 120.4(5) C(2)-C(4)-N(3) 110.8(4) N(4)-C(5)-N(5) 117.9(4) N(4)-C(5)-N(3) 124.6(5) N(5)-C(5)-N(3) 117.6(5) O(1)-C(7)-N(2) 109.0(5) O(1)-C(7)-C(8) 108.2(4) 169

Table D-3. Bond lengths [Å] and angles [°] for IV-2 (continued). ______

N(2)-C(7)-C(8) 113.7(4) O(3)-C(8)-C(7) 109.1(4) O(3)-C(8)-C(9) 113.6(4) C(7)-C(8)-C(9) 101.4(4) O(4)-C(9)-C(10) 108.9(5) O(4)-C(9)-C(8) 109.3(4) C(10)-C(9)-C(8) 103.3(4) O(1)-C(10)-C(11) 109.1(4) O(1)-C(10)-C(9) 105.5(4) C(11)-C(10)-C(9) 113.5(5) O(5)-C(11)-C(10) 110.2(4) ______

Symmetry transformations used to generate equivalent atoms:

Table D-4. Anisotropic displacement parameters (Å2x 103) for IV-2. The anisotropic displacement factor exponent takes the form: -2π2[ h2 a*2U11 + ... + 2 h k a* b* U12 ]

______

U11 U22 U33 U23 U13 U12

______

I(1) 12(1) 12(1) 16(1) 1(1) -2(1) 0(1) O(1) 12(2) 18(2) 9(2) 2(2) -1(1) -1(1) O(2) 20(2) 18(2) 9(2) 0(1) -2(2) 0(2) O(3) 12(2) 18(2) 14(2) 0(2) -2(2) 3(2) O(4) 26(2) 16(2) 12(2) -2(2) -3(2) 6(2) O(5) 20(2) 23(2) 14(2) -5(2) -4(2) 7(2) N(1) 8(2) 15(2) 9(2) -1(2) -1(2) 2(2) N(2) 13(2) 8(2) 13(2) 0(2) 2(2) -2(2) N(3) 14(2) 12(2) 10(2) -5(2) -2(2) 0(2) N(4) 11(2) 12(2) 10(2) -2(2) -1(2) -1(1) N(5) 33(3) 13(2) 7(2) -2(2) -3(2) -3(2) C(1) 12(2) 12(2) 10(2) 4(2) 1(2) 0(2) C(2) 6(2) 12(2) 9(2) 4(2) 4(2) 0(2) C(3) 9(2) 15(2) 10(2) -5(2) -1(2) -5(2) C(4) 8(2) 17(3) 11(2) 2(2) -1(2) -1(2) C(5) 9(2) 17(3) 9(2) -3(2) 0(2) 1(2) 170

Table D-4. Anisotropic displacement parameters (Å2x 103) for IV-2. The anisotropic displacement factor exponent takes the form: -2π2[ h2 a*2U11 + ... + 2 h k a* b* U12 ] (continued) ______C(6) 18(3) 11(2) 8(2) 1(2) 1(2) 2(2) C(7) 18(2) 7(2) 10(2) 0(2) -3(2) 2(2) C(8) 9(2) 18(2) 12(2) 4(2) 4(2) 2(2) C(9) 20(3) 12(2) 10(2) -1(2) 2(2) 5(2) C(10) 14(2) 19(3) 7(2) 1(2) 0(2) 3(2) C(11) 16(3) 16(3) 11(2) -1(2) 0(2) 0(2) C(12) 34(3) 13(2) 13(2) -7(2) -4(2) 6(2) ______

Table D-5. Hydrogen coordinates (x 104) and isotropic displacement parameters (Å2x 10 3) for IV-2. ______

x y z U(eq)

______

H(3) 670 6172 1636 22 H(4) 4206 7786 2303 27 H(5) 8618 5122 1217 29 H(5A) 5536 2534 1163 21 H(5B) 5579 1834 435 21 H(1) 3993 7422 -464 14 H(6A) 2423 6276 -1918 19 H(6B) 4366 5774 -2187 19 H(6C) 4243 7009 -1952 19 H(7) 4171 7404 960 14 H(13) 3101 5331 1498 15 H(9) 4043 5723 2765 17 H(8) 7137 6701 2429 16 H(10A) 7394 4897 2685 17 H(10B) 6236 4584 1919 17 H(12A) 6416 1776 -705 30 H(12B) 5358 2140 -1483 30 H(12C) 4205 1661 -764 30 ______

171

APPENDIX E.

CRYSTAL STRUCTURE DATA FOR C6H7BrCl2N2O2 (IV-3)

Table E-1. Crystal data and structure refinement for C6H7BrCl2N2O2. Empirical formula C6 H7 Br Cl2 N2 O2 Formula weight 289.95 Temperature 100(2) K Wavelength 0.71073 Å Crystal system Monoclinic Space group P2(1)/n Unit cell dimensions a = 5.774(3) Å α= 90°. b = 11.770(6) Å β= 97.015(8)°. c = 14.846(7) Å γ = 90°. Volume 1001.4(8) Å3 Z 4 Density (calculated) 1.923 Mg/m3 Absorption coefficient 4.607 mm-1 F(000) 568 Crystal size 0.15 x 0.08 x 0.05 mm3 Theta range for data collection 2.21 to 28.42°. Index ranges -7<=h<=7, -15<=k<=15, -19<=l<=19 Reflections collected 8473 Independent reflections 2374 [R(int) = 0.0374] Completeness to theta = 28.42° 94.0 % Absorption correction Semi-empirical from equivalents Max. and min. transmission 0.8024 and 0.5873 Refinement method Full-matrix least-squares on F2 Data / restraints / parameters 2374 / 0 / 120 Goodness-of-fit on F2 1.044 Final R indices [I>2sigma(I)] R1 = 0.0281, wR2 = 0.0650 R indices (all data) R1 = 0.0362, wR2 = 0.0672 Largest diff. peak and hole 1.233 and -0.360 e.Å-3

172

Table E-2. Atomic coordinates (x 104) and equivalent isotropic displacement parameters (Å2x 103) for IV-3. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor. ______

x y z U(eq)

______

Br(1) 2579(1) 1301(1) 2381(1) 17(1) Cl(1) 1397(1) 4221(1) 10979(1) 19(1) Cl(2) 3553(1) 3320(1) 8964(1) 18(1) O(1) 3033(3) 287(1) 7616(1) 20(1) O(2) 3681(3) 366(1) 9140(1) 20(1) N(2) -1304(3) 2422(2) 10396(1) 15(1) C(6) 2545(4) 591(2) 8424(2) 15(1) C(5) 307(4) 1259(2) 8336(2) 15(1) N(1) 48(3) 1870(2) 9172(1) 14(1) C(3) 1363(4) 2797(2) 9501(2) 14(1) C(4) -2772(4) 2502(2) 11133(2) 21(1) C(2) 526(4) 3137(2) 10265(2) 15(1) C(1) -1551(4) 1663(2) 9727(2) 15(1) ______

Table E-3. Bond lengths [Å] and angles [°] for IV-3. ______

Cl(1)-C(2) 1.694(2) Cl(2)-C(3) 1.691(2) O(1)-C(6) 1.314(3) O(2)-C(6) 1.208(3) N(2)-C(1) 1.331(3) N(2)-C(2) 1.383(3) N(2)-C(4) 1.467(3) C(6)-C(5) 1.505(3) C(5)-N(1) 1.458(3) N(1)-C(1) 1.333(3) N(1)-C(3) 1.384(3) C(3)-C(2) 1.347(3)

C(1)-N(2)-C(2) 108.54(19) C(1)-N(2)-C(4) 125.5(2) C(2)-N(2)-C(4) 126.0(2) O(2)-C(6)-O(1) 126.1(2) O(2)-C(6)-C(5) 123.9(2) 173

Table E-3. Bond lengths [Å] and angles [°] for IV-3 (continued). ______O(1)-C(6)-C(5) 109.92(19) N(1)-C(5)-C(6) 111.13(18) C(1)-N(1)-C(3) 108.52(19) C(1)-N(1)-C(5) 125.94(19) C(3)-N(1)-C(5) 125.49(19) C(2)-C(3)-N(1) 107.1(2) C(2)-C(3)-Cl(2) 130.89(18) N(1)-C(3)-Cl(2) 122.03(17) C(3)-C(2)-N(2) 107.23(19) C(3)-C(2)-Cl(1) 129.82(18) N(2)-C(2)-Cl(1) 122.95(17) N(2)-C(1)-N(1) 108.6(2) ______

Symmetry transformations used to generate equivalent atoms:

Table E-4. Anisotropic displacement parameters (Å2x 103) for IV-3. The anisotropic displacement factor exponent takes the form: -2π2[ h2 a*2U11 + ... + 2 h k a* b* U12 ]

______

U11 U22 U33 U23 U13 U12

______

Br(1) 13(1) 14(1) 24(1) -1(1) 2(1) 0(1) Cl(1) 21(1) 16(1) 18(1) -4(1) 0(1) -1(1) Cl(2) 16(1) 19(1) 20(1) 2(1) 4(1) -3(1) O(1) 18(1) 22(1) 19(1) 1(1) 2(1) 7(1) O(2) 18(1) 22(1) 20(1) -1(1) -2(1) 4(1) N(2) 11(1) 17(1) 16(1) 2(1) 3(1) 1(1) C(6) 13(1) 12(1) 20(1) 0(1) 1(1) -3(1) C(5) 15(1) 16(1) 15(1) -3(1) -1(1) 1(1) N(1) 12(1) 13(1) 16(1) -1(1) 0(1) -1(1) C(3) 13(1) 12(1) 16(1) 2(1) 0(1) 0(1) C(4) 17(1) 29(1) 18(1) 3(1) 5(1) 3(1) C(2) 12(1) 14(1) 17(1) 2(1) -2(1) 0(1) C(1) 12(1) 15(1) 19(1) 2(1) -1(1) -1(1) ______

174

Table E-5. Hydrogen coordinates (x 104) and isotropic displacement parameters (Å2x 10 3) for IV-3. ______

x y z U(eq)

______

H(1) 4241 -117 7671 30 H(5A) 296 1808 7830 19 H(5B) -1030 736 8192 19 H(4A) -4072 1965 11022 32 H(4B) -3386 3276 11159 32 H(4C) -1840 2318 11711 32 H(1A) -2678 1071 9657 18 ______

175

APPENDIX F.

CRYSTAL STRUCTURE DATA FOR C5H7I3N2 (IV-4)

Table F-1. Crystal data and structure refinement for C5H7I3N2. Empirical formula C5 H7 I3 N2 Formula weight 475.83 Temperature 100(2) K Wavelength 0.71073 Å Crystal system Tetragonal Space group I-42d Unit cell dimensions a = 16.289(3) Å α= 90°. b = 16.289(3) Å β= 90°. c = 8.0737(18) Å γ = 90°. Volume 2142.2(7) Å3 Z 8 Density (calculated) 2.951 Mg/m3 Absorption coefficient 8.698 mm-1 F(000) 1680 Crystal size 0.11 x 0.10 x 0.05 mm3 Theta range for data collection 2.50 to 28.22°. Index ranges -20<=h<=21, -21<=k<=21, -10<=l<=10 Reflections collected 9024 Independent reflections 1300 [R(int) = 0.0504] Completeness to theta = 28.22° 98.8 % Absorption correction Semi-empirical from equivalents Max. and min. transmission 0.647 and 0.4314 Refinement method Full-matrix least-squares on F2 Data / restraints / parameters 1300 / 0 / 48 Goodness-of-fit on F2 1.163 Final R indices [I>2sigma(I)] R1 = 0.0212, wR2 = 0.0453 R indices (all data) R1 = 0.0224, wR2 = 0.0456 Absolute structure parameter 0.07(7) Largest diff. peak and hole 0.724 and -0.648 e.Å-3

176

Table F-2. Atomic coordinates (x 104) and equivalent isotropic displacement parameters (Å2x 103) for IV-4. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor. ______

x y z U(eq)

______

I(1) -597(1) 1426(1) 7290(1) 15(1) I(2) 840(1) 2500 1250 14(1) N(1) 1224(2) 1887(2) 6752(5) 13(1) C(1) 1704(4) 2500 6250 17(1) C(2) 416(3) 2117(3) 6585(6) 15(1) C(3) 1515(3) 1105(3) 7448(6) 20(1) ______

Table F-3. Bond lengths [Å] and angles [°] for IV-4. ______

I(1)-C(2) 2.077(5) N(1)-C(1) 1.331(6) N(1)-C(2) 1.375(6) N(1)-C(3) 1.472(6) C(1)-N(1)#1 1.331(6) C(2)-C(2)#1 1.359(10)

C(1)-N(1)-C(2) 109.2(4) C(1)-N(1)-C(3) 125.2(4) C(2)-N(1)-C(3) 125.6(4) N(1)-C(1)-N(1)#1 108.1(6) C(2)#1-C(2)-N(1) 106.8(3) C(2)#1-C(2)-I(1) 127.32(14) N(1)-C(2)-I(1) 125.9(4) ______

Symmetry transformations used to generate equivalent atoms: #1 x+0,-y+1/2,-z+5/4

177

Table F-4. Anisotropic displacement parameters (Å2x 103) for IV-4. The anisotropic displacement factor exponent takes the form: -2π2[ h2 a*2U11 + ... + 2 h k a* b* U12 ]

______

U11 U22 U33 U23 U13 U12

______

I(1) 16(1) 15(1) 14(1) 1(1) 1(1) -4(1) I(2) 12(1) 12(1) 18(1) 2(1) 0 0 N(1) 12(2) 15(2) 13(2) 3(2) 1(2) 2(2) C(1) 14(3) 26(4) 12(3) -2(3) 0 0 C(2) 12(2) 24(3) 9(2) -3(2) 3(2) 1(2) C(3) 21(3) 21(3) 18(3) 5(2) 4(2) 10(2) ______

Table F-5. Hydrogen coordinates (x 104) and isotropic displacement parameters (Å2x 10 3) for IV-4. ______

x y z U(eq)

______

H(1) 2287 2500 6250 20 H(3A) 2109 1056 7272 30 H(3B) 1234 648 6898 30 H(3C) 1398 1090 8638 30

178

APPENDIX G.

CRYSTAL STRUCTURE DATA FOR C7H10I2N2O3 (IV-5)

Table G-1. Crystal data and structure refinement for C7H10I2N2O3. Empirical formula C7 H10 I2 N2 O3 Formula weight 423.97 Temperature 100(2) K Wavelength 0.71073 Å Crystal system Orthorhombic Space group Pnna Unit cell dimensions a = 7.486(4) Å α= 90°. b = 8.086(4) Å β= 90°. c = 20.000(10) Å γ = 90°. Volume 1210.7(11) Å3 Z 4 Density (calculated) 2.326 Mg/m3 Absorption coefficient 5.182 mm-1 F(000) 784 Crystal size 0.10 x 0.09 x 0.05 mm3 Theta range for data collection 2.04 to 28.35°. Index ranges -9<=h<=9, -10<=k<=10, -25<=l<=26 Reflections collected 9547 Independent reflections 1467 [R(int) = 0.0508] Completeness to theta = 28.35° 96.2 % Absorption correction None Refinement method Full-matrix least-squares on F2 Data / restraints / parameters 1467 / 0 / 68 Goodness-of-fit on F2 1.103 Final R indices [I>2sigma(I)] R1 = 0.0299, wR2 = 0.0648 R indices (all data) R1 = 0.0403, wR2 = 0.0693 Largest diff. peak and hole 1.113 and -1.063 e.Å-3

179

Table G-2. Atomic coordinates (x 104) and equivalent isotropic displacement parameters (Å2x 103) for IV-5. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor. ______

x y z U(eq)

______

I(1) 1858(1) 2593(1) 4452(1) 16(1) O(2) 1228(4) 721(3) 3337(1) 25(1) O(3) 8901(6) 2500 2500 30(1) N(1) 2132(4) 3710(4) 5916(2) 16(1) C(1) 2500 5000 6304(3) 16(1) C(3) 2273(5) 4187(4) 5252(2) 16(1) C(4) 1788(5) 2040(5) 6163(2) 22(1) C(5) 2500 0 3047(3) 27(2) C(6) 2500 0 2290(4) 83(4) ______

Table G-3. Bond lengths [Å] and angles [°] for IV-5. ______

I(1)-C(3) 2.078(4) O(2)-C(5) 1.258(4) N(1)-C(1) 1.329(4) N(1)-C(3) 1.388(5) N(1)-C(4) 1.460(5) C(1)-N(1) 1.329(4) C(3)-C(3) 1.358(7) C(5)-O(2) 1.258(4) C(5)-C(6) 1.515(10)

C(1)-N(1)-C(3) 108.9(3) C(1)-N(1)-C(4) 124.5(4) C(3)-N(1)-C(4) 126.4(3) N(1)-C(1)-N(1) 108.7(5) C(3)-C(3)-N(1) 106.8(2) C(3)-C(3)-I(1) 129.66(11) N(1)-C(3)-I(1) 123.6(3) O(2)-C(5)-O(2) 125.2(6)

180

Table G-3. Bond lengths [Å] and angles [°] for IV-5 (continued). ______O(2)-C(5)-C(6) 117.4(3) O(2)-C(5)-C(6) 117.4(3) ______Symmetry transformations used to generate equivalent atoms: #1 -x+1/2,-y+1,z #2 -x+1/2,-y,z

Table G-4. Anisotropic displacement parameters (Å2x 103) for IV-5. The anisotropic displacement factor exponent takes the form: -2π2[ h2 a*2U11 + ... + 2 h k a* b* U12 ]

______

U11 U22 U33 U23 U13 U12

______

I(1) 15(1) 17(1) 18(1) -3(1) 0(1) 1(1) O(2) 29(2) 25(2) 21(2) -1(1) 1(1) -1(1) O(3) 23(2) 41(3) 28(2) 2(2) 0 0 N(1) 14(2) 16(2) 18(2) 2(1) 0(1) -1(1) C(1) 17(3) 19(3) 12(3) 0 0 4(2) C(3) 11(2) 17(2) 19(2) -2(2) 0(2) 3(2) C(4) 26(2) 13(2) 26(2) 8(2) -2(2) -1(2) C(5) 47(4) 14(3) 20(3) 0 0 4(3) C(6) 106(8) 117(9) 24(4) 0 0 90(7) ______

Table G-5. Hydrogen coordinates (x 104) and isotropic displacement parameters (Å2x 10 3) for IV-5. ______

x y z U(eq)

______

H(1) 2500 5000 6779 19 H(4A) 2838 1345 6082 33 H(4B) 755 1573 5928 33 H(4C) 1539 2082 6644 33 H(6A) 3688 310 2126 124 H(6B) 1616 798 2126 124 H(6C) 2196 -1107 2126 124 181

APPENDIX H.

CRYSTAL STRUCTURE DATA FOR C24H27Br3Cl6N6O (IV-6)

Table H-1. Crystal data and structure refinement for C24H27Br3Cl6N6O. Empirical formula C24 H27 Br3 Cl6 N6 O Formula weight 867.95 Temperature 100(2) K Wavelength 0.71073 Å Crystal system Triclinic Space group P-1 Unit cell dimensions a = 10.664(4) Å α= 71.150(6)°. b = 11.149(4) Å β= 76.718(6)°. c = 15.404(6) Å γ= 68.310(6)°. Volume 1597.8(10) Å3 Z 2 Density (calculated) 1.804 Mg/m3 Absorption coefficient 4.320 mm-1 F(000) 856 Crystal size 0.14 x 0.09 x 0.05 mm3 Theta range for data collection 1.41 to 28.29°. Index ranges -13<=h<=14, -14<=k<=14, -20<=l<=19 Reflections collected 13584 Independent reflections 7145 [R(int) = 0.0314] Completeness to theta = 28.29° 90.2 % Absorption correction Semi-empirical from equivalents Max. and min. transmission 0.8130 and 0.5506 Refinement method Full-matrix least-squares on F2 Data / restraints / parameters 7145 / 0 / 367 Goodness-of-fit on F2 1.017 Final R indices [I>2sigma(I)] R1 = 0.0500, wR2 = 0.1027 R indices (all data) R1 = 0.0772, wR2 = 0.1141 Largest diff. peak and hole 0.853 and -0.711 e.Å-3

182

Table H-2. Atomic coordinates (x 104) and equivalent isotropic displacement parameters (Å2x 103) for IV-6. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor. ______

x y z U(eq)

______

Br(1) 4949(1) 1791(1) 2840(1) 30(1) Br(2) 1071(1) 6785(1) 5415(1) 32(1) Br(3) 1543(1) 7179(1) 52(1) 34(1) Cl(1) 4285(1) 6201(1) 5650(1) 26(1) Cl(2) 6532(1) 3361(1) 6969(1) 40(1) Cl(3) 11854(1) 11973(1) 2615(1) 27(1) Cl(4) 9953(1) 14948(1) 1278(1) 32(1) Cl(5) 5814(2) 10897(2) -1335(1) 47(1) Cl(6) 8065(1) 7834(2) -288(1) 38(1) N(1) 6624(4) 6794(3) 5352(3) 20(1) N(2) 7965(4) 5070(4) 6217(3) 23(1) N(3) 9365(4) 11690(4) 2870(3) 22(1) N(4) 8138(4) 13598(4) 2082(3) 25(1) N(5) 7532(4) 9329(4) 904(3) 27(1) N(6) 6108(4) 11243(4) 264(3) 34(1) C(1) 7127(4) 8546(4) 4008(3) 19(1) C(2) 7908(4) 9249(4) 4118(3) 21(1) C(3) 8898(4) 9564(4) 3396(3) 19(1) C(4) 9098(4) 9230(4) 2569(3) 21(1) C(5) 8273(4) 8565(4) 2458(3) 22(1) C(6) 7309(4) 8204(4) 3174(3) 22(1) C(7) 6050(4) 8169(4) 4770(3) 23(1) C(8) 7712(5) 9661(5) 4994(3) 24(1) C(9) 9745(5) 10323(4) 3499(3) 24(1) C(10) 10167(5) 9578(5) 1787(4) 30(1) C(11) 8450(5) 8246(5) 1547(3) 27(1) C(12) 6412(5) 7475(6) 3086(4) 35(1) C(13) 7862(4) 6265(4) 5627(3) 21(1) C(14) 5918(4) 5898(4) 5791(3) 23(1) C(15) 6765(4) 4817(4) 6316(3) 24(1) C(16) 9190(5) 4152(5) 6635(4) 32(1) C(17) 8112(5) 12410(5) 2635(3) 24(1) C(18) 10201(5) 12465(4) 2468(3) 22(1) C(19) 9444(5) 13640(5) 1972(3) 26(1) C(20) 6976(5) 14641(5) 1657(4) 34(1)

183

Table H-2. Atomic coordinates (x 104) and equivalent isotropic displacement parameters (Å2x 103) for IV-6. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor (continued). ______

C(21) 6789(5) 10557(5) 993(4) 28(1) C(22) 6467(5) 10429(6) -323(4) 33(1) C(23) 7348(5) 9251(5) 65(3) 29(1) C(24) 5232(6) 12644(5) 107(5) 45(2) O(1) 6344(5) 5044(5) 8424(4) 77(2) ______

Table H-3. Bond lengths [Å] and angles [°] for IV-6. ______

Cl(1)-C(14) 1.698(5) Cl(2)-C(15) 1.684(5) Cl(3)-C(18) 1.688(5) Cl(4)-C(19) 1.695(5) Cl(5)-C(22) 1.700(5) Cl(6)-C(23) 1.688(5) N(1)-C(13) 1.338(6) N(1)-C(14) 1.382(5) N(1)-C(7) 1.483(6) N(2)-C(13) 1.330(6) N(2)-C(15) 1.375(6) N(2)-C(16) 1.463(6) N(3)-C(17) 1.342(6) N(3)-C(18) 1.377(6) N(3)-C(9) 1.482(6) N(4)-C(17) 1.330(6) N(4)-C(19) 1.380(6) N(4)-C(20) 1.460(6) N(5)-C(21) 1.338(6) N(5)-C(23) 1.385(6) N(5)-C(11) 1.482(6) N(6)-C(21) 1.336(6) N(6)-C(22) 1.378(7) N(6)-C(24) 1.467(7) C(1)-C(2) 1.403(6) C(1)-C(6) 1.408(6) C(1)-C(7) 1.513(6) C(2)-C(3) 1.399(6) C(2)-C(8) 1.507(6) C(3)-C(4) 1.390(6) 184

Table H-3. Bond lengths [Å] and angles [°] for IV-6 (continued). ______

C(3)-C(9) 1.508(6) C(4)-C(5) 1.409(6) C(4)-C(10) 1.515(6) C(5)-C(6) 1.391(6) C(5)-C(11) 1.510(7) C(6)-C(12) 1.516(6) C(14)-C(15) 1.350(6) C(18)-C(19) 1.344(7) C(22)-C(23) 1.343(7)

C(13)-N(1)-C(14) 108.1(4) C(13)-N(1)-C(7) 125.9(4) C(14)-N(1)-C(7) 125.6(4) C(13)-N(2)-C(15) 108.5(4) C(13)-N(2)-C(16) 125.1(4) C(15)-N(2)-C(16) 126.3(4) C(17)-N(3)-C(18) 108.3(4) C(17)-N(3)-C(9) 125.1(4) C(18)-N(3)-C(9) 126.5(4) C(17)-N(4)-C(19) 108.0(4) C(17)-N(4)-C(20) 124.7(4) C(19)-N(4)-C(20) 127.2(4) C(21)-N(5)-C(23) 107.6(4) C(21)-N(5)-C(11) 127.5(4) C(23)-N(5)-C(11) 124.8(4) C(21)-N(6)-C(22) 107.5(4) C(21)-N(6)-C(24) 124.7(5) C(22)-N(6)-C(24) 127.7(5) C(2)-C(1)-C(6) 120.5(4) C(2)-C(1)-C(7) 121.0(4) C(6)-C(1)-C(7) 118.5(4) C(3)-C(2)-C(1) 118.8(4) C(3)-C(2)-C(8) 119.5(4) C(1)-C(2)-C(8) 121.7(4) C(4)-C(3)-C(2) 121.6(4) C(4)-C(3)-C(9) 118.7(4) C(2)-C(3)-C(9) 119.7(4) C(3)-C(4)-C(5) 118.8(4) C(3)-C(4)-C(10) 121.5(4) C(5)-C(4)-C(10) 119.7(4) C(6)-C(5)-C(4) 120.8(4) C(6)-C(5)-C(11) 120.2(4) C(4)-C(5)-C(11) 119.0(4) 185

Table H-3. Bond lengths [Å] and angles [°] for IV-6 (continued). ______

C(5)-C(6)-C(1) 119.4(4) C(5)-C(6)-C(12) 122.4(4) C(1)-C(6)-C(12) 118.1(4) N(1)-C(7)-C(1) 110.2(3) N(3)-C(9)-C(3) 109.6(4) N(5)-C(11)-C(5) 111.7(4) N(2)-C(13)-N(1) 108.8(4) C(15)-C(14)-N(1) 107.1(4) C(15)-C(14)-Cl(1) 129.7(4) N(1)-C(14)-Cl(1) 123.3(3) C(14)-C(15)-N(2) 107.5(4) C(14)-C(15)-Cl(2) 129.9(4) N(2)-C(15)-Cl(2) 122.6(3) N(4)-C(17)-N(3) 108.7(4) C(19)-C(18)-N(3) 107.1(4) C(19)-C(18)-Cl(3) 128.7(4) N(3)-C(18)-Cl(3) 124.3(4) C(18)-C(19)-N(4) 107.9(4) C(18)-C(19)-Cl(4) 128.2(4) N(4)-C(19)-Cl(4) 123.9(4) N(6)-C(21)-N(5) 109.5(5) C(23)-C(22)-N(6) 108.2(4) C(23)-C(22)-Cl(5) 128.5(5) N(6)-C(22)-Cl(5) 123.3(4) C(22)-C(23)-N(5) 107.3(4) C(22)-C(23)-Cl(6) 130.2(4) N(5)-C(23)-Cl(6) 122.4(4) ______

Symmetry transformations used to generate equivalent atoms:

Table H-4. Anisotropic displacement parameters (Å2x 103) for IV-6. The anisotropic displacement factor exponent takes the form: -2π2[ h2 a*2U11 + ... + 2 h k a* b* U12 ]

______

U11 U22 U33 U23 U13 U12

______

Br(1) 23(1) 32(1) 36(1) -7(1) -5(1) -10(1) 186

Table H-4. Anisotropic displacement parameters (Å2x 103) for IV-6. The anisotropic displacement factor exponent takes the form: -2π2[ h2 a*2U11 + ... + 2 h k a* b* U12 ] (continued). ______Br(2) 22(1) 38(1) 45(1) -21(1) -4(1) -12(1) Br(3) 31(1) 33(1) 42(1) -7(1) -3(1) -16(1) Cl(1) 21(1) 24(1) 39(1) -8(1) -4(1) -12(1) Cl(2) 37(1) 23(1) 57(1) 6(1) -10(1) -17(1) Cl(3) 25(1) 32(1) 29(1) -6(1) -5(1) -17(1) Cl(4) 41(1) 24(1) 35(1) -2(1) -7(1) -19(1) Cl(5) 44(1) 69(1) 31(1) 11(1) -17(1) -36(1) Cl(6) 37(1) 56(1) 32(1) -20(1) -3(1) -20(1) N(1) 20(2) 17(2) 27(2) -5(2) -2(2) -11(2) N(2) 20(2) 21(2) 29(2) -4(2) -8(2) -7(2) N(3) 26(2) 19(2) 27(2) -5(2) -8(2) -11(2)

N(4) 30(2) 18(2) 31(2) -4(2) -6(2) -12(2) N(5) 28(2) 30(2) 28(2) -5(2) -7(2) -17(2) N(6) 37(2) 30(2) 39(3) 6(2) -15(2) -21(2) C(1) 18(2) 16(2) 25(2) -3(2) -7(2) -5(2) C(2) 25(2) 15(2) 26(2) -1(2) -13(2) -6(2) C(3) 18(2) 15(2) 23(2) -2(2) -6(2) -6(2) C(4) 21(2) 17(2) 24(2) 1(2) -9(2) -7(2) C(5) 19(2) 17(2) 32(3) -6(2) -8(2) -4(2) C(6) 22(2) 17(2) 28(3) -1(2) -11(2) -9(2) C(7) 21(2) 20(2) 26(3) -3(2) -5(2) -7(2) C(8) 23(2) 22(2) 31(3) -9(2) -5(2) -9(2) C(9) 25(2) 22(2) 29(3) -2(2) -7(2) -13(2) C(10) 29(3) 30(3) 34(3) -9(2) -3(2) -15(2) C(11) 31(3) 24(2) 30(3) -6(2) -12(2) -11(2) C(12) 39(3) 46(3) 32(3) -12(2) -7(2) -26(3) C(13) 22(2) 16(2) 29(3) -6(2) -6(2) -10(2) C(14) 21(2) 22(2) 31(3) -8(2) 0(2) -13(2) C(15) 20(2) 23(2) 32(3) -5(2) -6(2) -10(2) C(16) 29(3) 23(2) 40(3) 0(2) -16(2) -5(2) C(17) 24(2) 28(2) 29(3) -13(2) -4(2) -12(2) C(18) 24(2) 24(2) 24(2) -10(2) 0(2) -14(2) C(19) 30(3) 25(2) 30(3) -6(2) -4(2) -17(2) C(20) 28(3) 26(3) 48(3) -9(2) -11(2) -7(2) C(21) 31(3) 24(2) 32(3) -1(2) -12(2) -12(2) C(22) 30(3) 46(3) 27(3) 6(2) -9(2) -25(2) C(23) 32(3) 41(3) 22(3) -9(2) -3(2) -20(2) C(24) 36(3) 31(3) 66(4) 5(3) -24(3) -11(2) O(1) 84(4) 60(3) 91(4) 8(3) -37(3) -37(3)

187

Table H-5. Hydrogen coordinates (x 104) and isotropic displacement parameters (Å2x 10 3) for IV-6. ______

x y z U(eq)

______

H(7A) 5711 8808 5157 27 H(7B) 5274 8217 4495 27 H(8A) 8537 9192 5299 36 H(8B) 6941 9429 5409 36 H(8C) 7530 10629 4842 36 H(9A) 9592 10375 4146 29

H(9B) 10722 9849 3347 29 H(10A) 9732 10375 1318 44 H(10B) 10627 8827 1506 44 H(10C) 10835 9756 2029 44 H(11A) 9405 8115 1259 32 H(11B) 8258 7401 1661 32 H(12A) 5457 7964 3250 52 H(12B) 6651 6571 3503 52 H(12C) 6550 7418 2447 52 H(13) 8554 6675 5433 25 H(16A) 9874 4605 6487 47 H(16B) 9553 3363 6390 47 H(16C) 8963 3874 7307 47 H(17) 7335 12120 2832 29 H(20A) 6180 14333 1853 50 H(20B) 7186 14837 983 50 H(20C) 6781 15453 1852 50 H(21) 6752 10889 1497 34 H(24A) 5107 12945 661 68 H(24B) 4346 12724 -28 68 H(24C) 5660 13200 -418 68

188

APPENDIX I.

CRYSTAL STRUCTURE DATA FOR C30H34Ag3Cl6N6O15 (IV-7)

Table I-1. Crystal data and structure refinement for C30H34Ag3Cl6N6O15. Empirical formula C30 H34 Ag3 Cl6 N6 O15 Formula weight 1254.95 Temperature 100(2) K Wavelength 0.71073 Å Crystal system Monoclinic Space group P2(1)/n Unit cell dimensions a = 16.134(4) Å α= 90°. b = 12.574(3) Å β= 98.581(5)°. c = 22.900(6) Å γ = 90°. Volume 4594(2) Å3 Z 4 Density (calculated) 2.006 Mg/m3 Absorption coefficient 2.071 mm-1 F(000) 2730 Crystal size 0.14 x 0.12 x 0.05 mm3 Theta range for data collection 1.45 to 28.40°. Index ranges -21<=h<=21, -16<=k<=16, -29<=l<=30 Reflections collected 38954 Independent reflections 10781 [R(int) = 0.0595] Completeness to theta = 28.40° 93.5 % Absorption correction Semi-empirical from equivalents Max. and min. transmission 0.9035 and 0.7079 Refinement method Full-matrix least-squares on F2 Data / restraints / parameters 10781 / 0 / 550 Goodness-of-fit on F2 1.099 Final R indices [I>2sigma(I)] R1 = 0.0536, wR2 = 0.1091 R indices (all data) R1 = 0.0820, wR2 = 0.1207 Largest diff. peak and hole 1.311 and -1.002 e.Å-3

189

Table I-2. Atomic coordinates (x 104) and equivalent isotropic displacement parameters (Å2x 103) for IV-7. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor. ______

x y z U(eq)

______

C(17) 7901(3) 9056(4) 4153(3) 26(1) C(18) 8834(4) 9255(5) 4301(3) 36(2) Ag(3) 4588(1) 5769(1) 1128(1) 23(1) Ag(2) 6536(1) 6383(1) 3705(1) 23(1) Ag(1) 6284(1) 8712(1) 3511(1) 23(1) Cl(4) 9202(1) 6409(1) 2196(1) 24(1) Cl(1) 3015(1) 10347(1) 3681(1) 27(1) Cl(2) 3027(1) 9705(1) 2192(1) 26(1) Cl(5) 2602(1) 1982(1) 1303(1) 38(1) Cl(3) 10088(1) 6452(1) 3680(1) 33(1) Cl(6) 3507(1) 2654(1) 2744(1) 26(1) N(4) 8404(3) 6369(3) 3708(2) 20(1) O(6) 4169(2) 8056(3) 801(2) 29(1) C(26) 3253(3) 3005(4) 1544(2) 22(1) N(3) 7856(3) 6293(3) 2789(2) 18(1) C(21) 8722(3) 6352(4) 2811(2) 20(1) N(1) 4543(3) 9030(3) 2751(2) 17(1) C(9) 7282(3) 6157(4) 2225(2) 21(1) O(3) 5713(2) 6724(3) 4336(2) 29(1) C(1) 5202(3) 7558(4) 2271(2) 19(1) N(6) 3551(3) 3721(3) 1167(2) 21(1) N(2) 4511(3) 9381(4) 3670(2) 21(1) C(4) 5855(3) 5484(4) 2382(2) 19(1) C(2) 6054(3) 7386(4) 2241(2) 18(1) C(25) 4046(3) 4454(4) 1481(2) 20(1) O(5) 5251(2) 6984(3) 734(2) 28(1) C(5) 5011(3) 5667(4) 2429(2) 17(1) C(30) 5399(4) 8813(5) 518(3) 31(1) C(20) 9060(3) 6399(4) 3377(2) 21(1) C(7) 4829(3) 8669(4) 2207(2) 21(1) C(16) 4732(4) 9459(5) 4304(2) 27(1) C(14) 3762(3) 9717(4) 3356(2) 23(1) C(13) 5008(3) 8976(4) 3297(2) 20(1) C(8) 6656(3) 8297(4) 2169(2) 25(1) C(3) 6371(3) 6341(4) 2292(2) 20(1) C(29) 4892(3) 7885(4) 694(2) 23(1)

190

Table I-2. Atomic coordinates (x 104) and equivalent isotropic displacement parameters (Å2x 103) for IV-7. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor (continued). ______

N(5) 4059(3) 4176(3) 2050(2) 18(1) C(27) 3576(3) 3277(4) 2097(2) 21(1) C(15) 3774(3) 9508(4) 2785(2) 20(1) C(10) 6202(3) 4362(4) 2428(3) 25(1) C(6) 4679(3) 6696(4) 2361(2) 19(1) C(11) 4467(3) 4763(4) 2581(2) 18(1) C(19) 7658(3) 6317(4) 3344(2) 21(1) C(12) 3746(3) 6884(4) 2374(3) 25(1) C(22) 8511(3) 6435(5) 4351(2) 26(1) O(1) 7623(2) 8829(3) 3622(2) 29(1) O(2) 7451(3) 9136(4) 4553(2) 38(1) C(28) 3353(4) 3680(5) 524(2) 31(1) O(9) 7805(2) 8270(3) 5634(2) 32(1) O(8) 6234(2) 7358(3) 5514(2) 33(1) O(15) 1739(2) 9599(3) 1110(2) 35(1) O(4) 4740(3) 5673(3) 3843(2) 31(1) C(23) 4991(3) 6287(4) 4257(2) 24(1) C(24) 4419(4) 6522(6) 4708(3) 38(2) O(7) 4881(2) 7984(3) 6095(2) 31(1) O(11) 5868(3) 3990(3) 3817(2) 36(1) O(14) 3129(3) 9926(4) 520(2) 41(1) O(12) 6563(3) 1072(4) 4527(2) 44(1) O(13) 7073(3) 7123(4) 853(2) 41(1) O(10) 7490(3) 4141(4) 4415(2) 52(1) ______

Table I-3. Bond lengths [Å] and angles [°] for IV-7. ______

C(17)-O(2) 1.254(7) C(17)-O(1) 1.263(7) C(17)-C(18) 1.514(8) Ag(3)-C(25) 2.088(5) Ag(3)-O(5) 2.140(4) Ag(2)-C(19) 2.101(5) Ag(2)-O(3) 2.147(4) Ag(2)-Ag(1) 2.9810(10) Ag(1)-C(13) 2.070(5) Ag(1)-O(1) 2.143(4) Cl(4)-C(21) 1.706(5) 191

Table I-3. Bond lengths [Å] and angles [°] for IV-7 (continued). ______

Cl(1)-C(14) 1.702(5) Cl(2)-C(15) 1.694(5) Cl(5)-C(26) 1.701(5) Cl(3)-C(20) 1.701(5) Cl(6)-C(27) 1.695(5) N(4)-C(19) 1.360(6) N(4)-C(20) 1.392(6) N(4)-C(22) 1.459(7) O(6)-C(29) 1.245(6) C(26)-C(27) 1.339(7) C(26)-N(6) 1.381(7) N(3)-C(19) 1.357(6) N(3)-C(21) 1.392(6) N(3)-C(9) 1.482(6) C(21)-C(20) 1.331(7) N(1)-C(13) 1.361(7) N(1)-C(15) 1.391(6) N(1)-C(7) 1.463(6) C(9)-C(3) 1.520(7) O(3)-C(23) 1.276(7) C(1)-C(2) 1.404(7) C(1)-C(6) 1.407(7) C(1)-C(7) 1.519(7) N(6)-C(25) 1.354(6) N(6)-C(28) 1.461(7) N(2)-C(13) 1.354(7) N(2)-C(14) 1.378(7) N(2)-C(16) 1.448(7) C(4)-C(3) 1.395(7) C(4)-C(5) 1.402(7) C(4)-C(10) 1.516(7) C(2)-C(3) 1.408(7) C(2)-C(8) 1.528(7) C(25)-N(5) 1.346(6) O(5)-C(29) 1.269(6) C(5)-C(6) 1.400(7) C(5)-C(11) 1.507(7) C(30)-C(29) 1.515(7) C(14)-C(15) 1.336(7) N(5)-C(27) 1.387(6) N(5)-C(11) 1.488(6) C(6)-C(12) 1.528(7) O(4)-C(23) 1.243(6) 192

Table I-3. Bond lengths [Å] and angles [°] for IV-7 (continued). ______

C(23)-C(24) 1.514(8) O(2)-C(17)-O(1) 124.0(5) O(2)-C(17)-C(18) 119.1(5) O(1)-C(17)-C(18) 116.8(5) C(25)-Ag(3)-O(5) 173.10(17) C(19)-Ag(2)-O(3) 158.20(18) C(19)-Ag(2)-Ag(1) 94.95(14) O(3)-Ag(2)-Ag(1) 79.60(11) C(13)-Ag(1)-O(1) 165.10(18) C(13)-Ag(1)-Ag(2) 107.55(14) O(1)-Ag(1)-Ag(2) 86.40(11) C(19)-N(4)-C(20) 110.0(4) C(19)-N(4)-C(22) 125.6(4) C(20)-N(4)-C(22) 124.3(4) C(27)-C(26)-N(6) 107.6(5) C(27)-C(26)-Cl(5) 129.4(4) N(6)-C(26)-Cl(5) 123.0(4) C(19)-N(3)-C(21) 109.9(4) C(19)-N(3)-C(9) 128.0(4) C(21)-N(3)-C(9) 122.0(4) C(20)-C(21)-N(3) 107.5(4) C(20)-C(21)-Cl(4) 129.1(4) N(3)-C(21)-Cl(4) 123.3(4) C(13)-N(1)-C(15) 110.1(4) C(13)-N(1)-C(7) 124.4(4) C(15)-N(1)-C(7) 125.4(4) N(3)-C(9)-C(3) 112.5(4) C(23)-O(3)-Ag(2) 117.5(4) C(2)-C(1)-C(6) 120.1(5) C(2)-C(1)-C(7) 120.9(5) C(6)-C(1)-C(7) 119.0(4) C(25)-N(6)-C(26) 110.2(4) C(25)-N(6)-C(28) 125.6(5) C(26)-N(6)-C(28) 124.3(5) C(13)-N(2)-C(14) 110.1(4) C(13)-N(2)-C(16) 125.3(4) C(14)-N(2)-C(16) 124.6(4) C(3)-C(4)-C(5) 119.5(5) C(3)-C(4)-C(10) 120.5(5) C(5)-C(4)-C(10) 120.0(5) C(1)-C(2)-C(3) 119.1(5) C(1)-C(2)-C(8) 122.2(5)

193

Table I-3. Bond lengths [Å] and angles [°] for IV-7 (continued).

C(3)-C(2)-C(8) 118.7(4) N(5)-C(25)-N(6) 105.0(4) N(5)-C(25)-Ag(3) 129.3(4) N(6)-C(25)-Ag(3) 125.5(4) C(29)-O(5)-Ag(3) 114.6(3) C(6)-C(5)-C(4) 120.2(5) C(6)-C(5)-C(11) 119.8(4) C(4)-C(5)-C(11) 120.0(4) C(21)-C(20)-N(4) 107.2(4) C(21)-C(20)-Cl(3) 129.3(4) N(4)-C(20)-Cl(3) 123.5(4) N(1)-C(7)-C(1) 111.9(4) C(15)-C(14)-N(2) 108.0(5) C(15)-C(14)-Cl(1) 129.3(4) N(2)-C(14)-Cl(1) 122.5(4) N(2)-C(13)-N(1) 105.2(4) N(2)-C(13)-Ag(1) 124.9(4) N(1)-C(13)-Ag(1) 128.2(4) C(4)-C(3)-C(2) 121.0(5) C(4)-C(3)-C(9) 120.3(5) C(2)-C(3)-C(9) 118.6(5) O(6)-C(29)-O(5) 124.8(5) O(6)-C(29)-C(30) 118.6(5) O(5)-C(29)-C(30) 116.6(5) C(25)-N(5)-C(27) 111.0(4) C(25)-N(5)-C(11) 127.0(4) C(27)-N(5)-C(11) 121.8(4) C(26)-C(27)-N(5) 106.2(5) C(26)-C(27)-Cl(6) 129.9(4) N(5)-C(27)-Cl(6) 123.7(4) C(14)-C(15)-N(1) 106.5(4) C(14)-C(15)-Cl(2) 130.0(4) N(1)-C(15)-Cl(2) 123.5(4) C(5)-C(6)-C(1) 120.0(5) C(5)-C(6)-C(12) 120.1(5) C(1)-C(6)-C(12) 119.8(5) N(5)-C(11)-C(5) 112.8(4) N(3)-C(19)-N(4) 105.4(4) N(3)-C(19)-Ag(2) 135.0(4) N(4)-C(19)-Ag(2) 119.5(4) C(17)-O(1)-Ag(1) 109.7(3) O(4)-C(23)-O(3) 124.2(5) O(4)-C(23)-C(24) 118.7(5) O(3)-C(23)-C(24) 117.1(5) 194

Table I-4. Anisotropic displacement parameters (Å2x 103) for IV-7. The anisotropic displacement factor exponent takes the form: -2π2[ h2 a*2U11 + ... + 2 h k a* b* U12 ]

______

U11 U22 U33 U23 U13 U12

______

C(17) 20(3) 25(3) 34(3) 6(2) 3(2) 4(2) C(18) 22(3) 42(4) 45(4) -1(3) 7(3) -2(3) Ag(3) 20(1) 25(1) 25(1) 3(1) 6(1) -2(1) Ag(2) 14(1) 31(1) 26(1) 2(1) 8(1) 0(1) Ag(1) 14(1) 31(1) 25(1) -1(1) 2(1) 0(1) Cl(4) 15(1) 30(1) 29(1) 0(1) 10(1) -1(1) Cl(1) 19(1) 31(1) 33(1) -5(1) 9(1) 2(1) Cl(2) 18(1) 31(1) 29(1) -1(1) -2(1) 6(1) Cl(5) 46(1) 30(1) 35(1) -3(1) -3(1) -18(1) Cl(3) 11(1) 52(1) 36(1) -2(1) 1(1) -5(1) Cl(6) 26(1) 26(1) 27(1) 5(1) 5(1) -4(1) N(4) 11(2) 27(2) 21(2) 0(2) 2(2) 0(2) O(6) 16(2) 40(2) 34(2) 0(2) 10(2) -2(2) C(26) 22(3) 15(3) 27(3) -1(2) 4(2) 0(2) N(3) 11(2) 20(2) 21(2) -1(2) 3(2) -2(2) C(21) 11(2) 20(3) 29(3) -1(2) 7(2) 1(2) N(1) 15(2) 15(2) 22(2) -1(2) 3(2) 2(2) C(9) 14(3) 25(3) 23(3) -2(2) 5(2) 1(2) O(3) 23(2) 34(2) 31(2) -5(2) 9(2) -3(2) C(1) 14(3) 21(3) 20(3) -2(2) 4(2) 0(2) N(6) 21(2) 18(2) 24(2) 3(2) 4(2) 3(2) N(2) 15(2) 26(2) 24(2) -1(2) 5(2) 1(2) C(4) 16(3) 21(3) 19(3) 3(2) 2(2) 3(2) C(2) 12(2) 22(3) 20(3) -1(2) 2(2) 0(2) C(25) 14(3) 24(3) 22(3) -3(2) 3(2) 3(2) O(5) 23(2) 28(2) 35(2) 8(2) 9(2) 0(2) C(5) 11(2) 22(3) 19(3) 0(2) 2(2) 1(2) C(30) 24(3) 32(3) 39(4) 10(3) 6(3) -6(3) C(20) 10(2) 26(3) 26(3) -1(2) 1(2) 0(2) C(7) 15(3) 23(3) 25(3) 1(2) 2(2) 1(2) C(16) 23(3) 38(3) 20(3) 2(2) 4(2) -2(2) C(14) 15(3) 28(3) 27(3) 0(2) 7(2) 1(2) C(13) 17(3) 17(3) 28(3) 3(2) 4(2) -2(2) C(8) 19(3) 26(3) 31(3) -2(2) 8(2) 0(2) C(3) 12(2) 29(3) 18(3) -3(2) 2(2) 0(2)

195

Table I-4. Anisotropic displacement parameters (Å2x 103) for IV-7. The anisotropic displacement factor exponent takes the form: -2π2[ h2 a*2U11 + ... + 2 h k a* b* U12 ] (continued) ______C(29) 26(3) 23(3) 20(3) 0(2) 1(2) -2(2) N(5) 14(2) 20(2) 19(2) 1(2) 3(2) 0(2) C(27) 18(3) 19(3) 27(3) 3(2) 3(2) 1(2) C(15) 14(3) 23(3) 23(3) -1(2) 0(2) 1(2) C(10) 15(3) 29(3) 33(3) 3(2) 6(2) -1(2) C(6) 12(2) 26(3) 20(3) -2(2) 0(2) -1(2) C(11) 13(2) 21(3) 22(3) -1(2) 4(2) 1(2) C(19) 16(3) 23(3) 23(3) 4(2) 3(2) 0(2) C(12) 12(3) 25(3) 38(3) -5(2) 6(2) 1(2) C(22) 17(3) 36(3) 27(3) -3(3) 4(2) 5(2) O(1) 16(2) 42(2) 29(2) -3(2) 3(2) 0(2) O(2) 27(2) 57(3) 32(2) -2(2) 10(2) 5(2) C(28) 38(4) 32(3) 22(3) -6(3) 0(3) 3(3) O(9) 20(2) 40(2) 40(2) -5(2) 11(2) 0(2) O(8) 20(2) 40(2) 42(3) -7(2) 8(2) -4(2) O(15) 23(2) 44(3) 37(2) 3(2) 4(2) 6(2) O(4) 30(2) 36(2) 30(2) -6(2) 10(2) -3(2) C(23) 22(3) 23(3) 26(3) 2(2) 4(2) 3(2) C(24) 21(3) 59(4) 36(4) -9(3) 11(3) -10(3) O(7) 25(2) 38(2) 30(2) -4(2) 6(2) 1(2) O(11) 30(2) 44(3) 36(2) 6(2) 10(2) 0(2) O(14) 39(3) 44(3) 42(3) 2(2) 15(2) 5(2) O(12) 29(2) 60(3) 45(3) 12(2) 10(2) 13(2) O(13) 30(2) 58(3) 35(2) -1(2) 7(2) 4(2) O(10) 34(3) 57(3) 65(3) -4(3) 5(2) -4(2) ______

Table I-5. Hydrogen coordinates (x 104) and isotropic displacement parameters (Å2x 10 3) for IV-7. ______

x y z U(eq)

______

H(18A) 9130 8844 4033 54 H(18B) 9028 9037 4709 54 H(18C) 8948 10015 4257 54 H(9A) 7345 5428 2074 25 H(9B) 7443 6663 1930 25

196

Table I-5. Hydrogen coordinates (x 104) and isotropic displacement parameters (Å2x 10 3) for IV-7 (continued). ______H(30A) 5068 9467 513 47 H(30B) 5545 8686 123 47 H(30C) 5913 8888 803 47 H(7A) 5256 9170 2102 26 H(7B) 4350 8671 1882 26 H(16A) 5265 9093 4429 40 H(16B) 4292 9126 4495 40 H(16C) 4787 10209 4419 40 H(13) 4853 8221 3365 24 H(8A) 7188 8178 2428 37 H(8B) 6410 8970 2273 37 H(8C) 6757 8327 1757 37 H(10A) 6610 4277 2154 38 H(10B) 5743 3853 2327 38 H(10C) 6477 4230 2833 38 H(11A) 4814 4261 2846 22 H(11B) 4028 5048 2797 22 H(12A) 3432 6237 2246 38 H(12B) 3549 7470 2106 38 H(12C) 3660 7066 2776 38 H(22A) 7966 6564 4478 40 H(22B) 8894 7020 4485 40 H(22C) 8745 5765 4521 40 H(28A) 3628 4273 353 47 H(28B) 2745 3733 407 47 H(28C) 3553 3006 381 47 H(24A) 3961 6002 4668 57 H(24B) 4187 7239 4643 57 H(24C) 4739 6475 5106 57

197

APPENDIX J.

LIST OF ABBREVIATIONS

19-L 6-hydroxycoumarin-3-carboxylic acid

20-L 7-hydroxycoumarin-3-carboxylic acid

21-L 8-hydroxycoumarin-3-carboxylic acid

22-L coumarin-3-carboxylic acid

Å Angstrom

α crystallographic unit-cell angle between axes b and c

β crystallographic unit-cell angle between axes a and c

γ crystallographic unit-cell angle between axes a and b

δ scale (NMR) ppm

µM micromolar a crystallographic unit cell axis a

Ag2O silver oxide

AgClO4 silver pechlorate

AgNO3 silver nitrate

AgOAc silver acetate anal. Analysis azpy 2-phenylazopyridine 198

b crystallographic unit cell axis b

C Celsius c crystallographic unit cell axis c calcd. Calculated

CDCl3 deuterated chloroform

CH2Cl2 methylene chloride

CH3CN acetonitrile

CH3COOH acetic acid

CHCl3 chloroform cpd cyclopentane-1,1-dicarboxylate

CuCl2 copper dichloride d doublet (NMR spectra) d(GpG) deoxy-guanosine-phosphate-guanosine depe 1,2-bis(diethylphosphino)ethane

DMA dimethyl acetamide

DMF dimethylformamide

DMSO dimethylsulfoxide

DNA deoxyribonucleic acid dppe 1,2-bis(diphenylphosphino)ethane dppey cis-1,2-bis(diphenylphosphino)ethylene

ED50 concentration needed to cause 50% loss of protein synthesis

ESI-MS electro spray ionization mass spectrometry et. al. and others

199

Et2O diethyl ether

Et3N triethylamine

EtOH ethanol

F(000) scaling coefficient for structure factors

Fc calculated structure factor

Fo observed structure factor

Fac facial fbc 4-fluorobenzoate

GSH glutathione h hour

Hz hertz

HBr hydrobromic acid

HCl hydrochloric acid

IC50 concentration needed to inhibit 50% of cell proliferation

ID50 dose needed to inhibit 50% of cell proliferation idc iminodiacetate

ILS percent increased lifespan

K2CO3 potassium carbonate

LD10 dose needed to kill 10% of the animals tested

LD50 dose needed to kill 50% of the animals/cells tested

LiBr lithium bromide m multiplet

M molar

200

MeOH methanol mL milliliter mmol millimole

MTD maximum tolerated dose

N/A not attained

Na2SO4 sodium sulfate

NaCl sodium chloride

NH4OH ammonium hydroxide

NH4PF6 ammonium hexaflurophosphate

NHC N-heterocyclic carbene nm nanometer

NMR nuclear magnetic resonance

PBS phosphate buffered saline

Ph phenyl ppm parts per million q quartet (spectral)

R organic group

R, R residual based on Fo

RhCl3 rhodium trichloride

RT room temperature s singlet (spectrum)

SDS sodium dodecyl sulfate t triplet (spectrum)

THF tetrahydrofuran 201

TsCl para-toluenesulfonyl chloride

U temperature factor unsat. Unsaturated wR2 weighted residual based on I

202

APPENDIX K

APPROVAL FOR ANIMAL USE

203