Development of Non-Traditional Platinum Anticancer Agents: Trans-Platinum Planar Amine Compounds and Polynuclear Platinum Compounds

Virginia Commonwealth University VCU Scholars Compass

Theses and Dissertations Graduate School

2015

Development of Non-Traditional Platinum Anticancer Agents: trans-Platinum Planar Amine Compounds and Polynuclear Platinum Compounds

Daniel E. Lee Virginia Commonwealth University

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DEVELOPMENT OF NON-TRADITIONAL PLATINUM ANTICANCER AGENTS: TRANS- PLATINUM PLANAR AMINE COMPOUNDS AND POLYNUCLEAR PLATINUM COMPOUNDS

A Dissertation Submitted in the Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Chemistry at Virginia Commonwealth University.

Daniel E. Lee B. Sc in Chemistry, Temple University, 2007

Advisor: Nicholas P. Farrell, Professor, Department of Chemistry, College of Humanities and Sciences, Virginia Commonwealth University

Virginia Commonwealth University Richmond, VA February, 2015

ACKNOWLEDGEMENTS

What a journey it has been! It has been the amazing experience but also probably has been the most difficult time of my life so far. I have met many wonderful individuals throughout the graduate program and I would like to express my sincere gratitude for all the supports and contributions. First of all, I would like to thank Dr. Farrell for his guidance and support throughout the entire program. I would like to thank Samantha Tsotsoros and Sarah Spell (my dear friends, siblings, lab-mates, lunch buddies, and so on). It would not have been the same without you two.

I would like to thank Dr.Qu for all of your valuable advice and assistance with NMR. I would like to thank my committee members (Dr. Sidorov, Dr. Collinson, and Dr. Ryan) for all of the guidance and valuable comments. I would like to thank Erica Peterson, Vijay Menon (former biology collaborator) and Samantha Katner (current biology collaborator) for all of your assistance in biological side of the lab. I would like to thank A. Gerard Daniel for all of your inputs based on theoretical chemistry and modeling. I would also like to thank our futures of the lab, Jamie Beaton,

Wyatt Johnson, and Eric Ginsburg. Last but not least, I would like to thank my family for always being so faithful, supportive, and loving.

TABLE OF CONTENTS

Acknowledgements ...... ii

Table of Contents ...... iii

List of Figures ...... viii

List of Tables ...... xvii

Abstract ...... xxi

Chapter 1. Introduction ...... 2

1.1 Cancer ...... 2

1.2 Treatments ...... 3

1.3 Platinum-Based Chemotherapeutics ...... 3

1.4 Mechanism of Action of Cisplatin ...... 4

1.5 Cisplatin Resistance ...... 6

1.6 Nontraditional Platinum Compounds ...... 9

1.6.1 trans-Platinum Planar Amine (TPA) Compounds ...... 9

1.6.2 Polynuclear Platinum Compounds (PPC) ...... 13

1.6.3 Noncovalently Binding Cationic Polynuclear Platinum Compounds (PPC-NC) ... 15

1.7 Aim and Scope of This Thesis ...... 18

Chapter 2. Synthesis, Characterization, and Chemical Property of trans-Platinum

Planar Amine Compounds ...... 20

iii

2.1 Structure-Based Rational TPA Drug Design ...... 21

2.1.1 Effects of Planar Amines Carrier Ligands on Cytotoxicity ...... 21

2.1.2 Effects of Carboxylate Leaving Groups on Cytotoxicity and Cellular Accumulation

2.2 Synthesis and Characterization ...... 23

2.3 Evaluation of TPA Compounds by Chemical Techniques ...... 27

2.3.1 Experimental Condition ...... 28

2.3.2 Results ...... 29

2.4 Binding of TPA Compounds with DNA ...... 31

2.4.1 Experimental Conditions ...... 33

2.4.2 Results ...... 35

2.5 Contributions ...... 36

Chapter 3. Biological Effect of TPA Compounds ...... 38

3.1 Cytotoxicity, Cellular Accumulation, and Metabolic Stability of TPA Compounds ...... 39

3.1.1 Experimental ...... 39

3.1.2 Results ...... 41

3.2 Cell Cycle Effects of TPA Compounds ...... 45

3.2.1 Experimental ...... 45

3.2.2 Cell Cycle Effects ...... 45

3.3 Possible Cellular Targets Other Than DNA ...... 46

3.3.1 Ternary Topoisomerase I-DNA Interaction ...... 47

3.3.2 Zinc-Finger Proteins ...... 48 iv

3.3.3 Telomerase ...... 49

3.3.4 Nuclear Protein Positive Cofactor PC4...... 49

3.4 Conclusion ...... 50

3.5 Contributions ...... 51

Chapter 4. Synthesis and Characterization of Polynuclear Platinum Compounds ...... 53

4.1 Synthetic Considerations ...... 57

4.1.1 Synthetic Considerations of PPC Chloride Analogs (PPC-Cl) ...... 57

4.1.2 Synthetic Considerations of PPC-NC ...... 58

4.2 Synthesis and Characterization of PPC-Cl ...... 60

4.2.1 Synthesis of BBR3464 by New Approach...... 61

4.2.2 Synthesis of Tetraplatin-Cl ...... 62

4.3 Synthesis and Characterization of PPC-NC ...... 62

4.3.1 Synthesis of Monoplatin NC ...... 62

4.3.2 Synthesis of Diplatin NC, Triplatin NC and Tetraplatin NC ...... 63

4.4 Contributions ...... 66

Chapter 5. DNA Binding Profiles and Cellular Effects of Polynuclear Platinum

Compounds 68

5.1 Determination of Platinum Compound-DNA Interactions ...... 69

5.1.1 Background ...... 69

5.1.2 Experimental Conditions ...... 73

5.1.3 Results and Discussion ...... 74

5.2 Biological Activities of Polynuclear NC Compounds ...... 84

5.2.1 Experimental ...... 84

5.2.2 Cellular Accumulation of PPC-NC in HC116 Cells ...... 86

5.2.3 Growth Inhibition Effects of PPC-NC ...... 88

5.3 Interactions of PPC and Heparan Sulfate...... 89

5.3.1 Cellular Accumulation of PPC-NC in HSPG deficient Cells ...... 93

5.3.2 Interaction with D-glucosamine-3-sulfate by HSQC [1H, 15N] Spectroscopy and ITC

5.3.3 Colorimetric Assay to Quantify PPC-NC and Heparin Sulfate Interaction ...... 98

5.3.4 Effects of Heparin in DNA-Pt Binding ...... 103

5.4 Contributions ...... 107

Chapter 6. Other Contributions ...... 109

6.1 Hybrid Polynuclear Platinum Compounds ...... 109

6.1.1 Synthesis of Pentaplatin-Cl ...... 111

6.1.2 Active Metabolites of Novel PPC-Cl with Dual Antitumor Activity ...... 112

6.2 Synthesis of a Carboxylate Derivative of BBR3464 ...... 116

6.3 Synthesis of TPA Succinate Compound...... 118

6.3.1 Attempt to synthesize the succinate TPA by utilizing methylated succinate followed

by acid hydrolysis ...... 120

6.3.2 Synthesis of TPA-succinato compounds by using excess succinic acid ...... 122

6.4 Conjugation of NBD-Fluorophore ...... 123

6.4.1 Preparation of 6-NBD ...... 125

6.4.2 Attempt to Conjugate 6-NBD to PPC ...... 126

6.5 Synthesis of Fluorine-18 Labelled Cisplatin ...... 129

6.6 Platinum Recovery ...... 132

6.7 Contributions ...... 134

References ...... 135

vii

LIST OF FIGURES

FIGURE 1.1. THE ROLE OF GENES AND ENVIRONMENT IN THE DEVELOPMENT OF CANCER. A. THE

PERCENTAGE CONTRIBUTION OF GENETIC AND ENVIRONMENTAL FACTORS TO CANCER. B.

FAMILY RISK RATIOS FOR SELECTED CANCERS. C. PERCENTAGE CONTRIBUTION OF EACH

ENVIRONMENTAL FACTOR FROM REF. (1)...... 2

FIGURE 1.2. FDA APPROVED PLATINUM-BASED ANTICANCER AGENTS ...... 4

FIGURE 1.3. MECHANISM OF ACTION OF CISPLATIN ...... 5

FIGURE 1.4. FORMATION OF CISPLATIN ADDUCTS. FROM REF (23)...... 6

FIGURE 1.5.STRUCTURE OF SULFUR NUCLEOPHILES: CYSTEINE, METHIONINE, AND GLUTATHONE. . 7

FIGURE 1.6. RESISTANCE TO CISPLATIN AND CARBOPLATIN MEDIATED AFTER DNA BINDING.

FROM REF (10)...... 8

FIGURE 1.7. STRUCTURES OF TRANS-PTTZ, TRANS-[PTNH3(THIAZOLE)CL2], AND BBR3464,

4+ [{TRANS-PTCL(NH3)2}2(µ-TRANS-PT(NH3)2{NH2(CH2)6NH2}2)] ...... 9

FIGURE 1.8. STRUCTURE OF CISPLATIN AND TRANSPLATIN ...... 9

FIGURE 1.9. CROSS-LINKS FORMED BY TRANS-PTTZ. FROM REF (82) ...... 11

FIGURE 1.10. RATIONAL DESIGN OF TPA COMPOUNDS...... 12

FIGURE 1.11. STRUCTURE OF BBR3464...... 13

FIGURE 1.12. STRUCTURES FROM MOLECULAR MODELING OF THE MAJOR DNA ADDUCTS OF

BBR3464. FROM REF (82)...... 14

FIGURE 1.13. STRUCTURES OF TRINUCLEAR PLATINUM COMPOUNDS...... 16

FIGURE 2.1. GENERAL STRUCTURE OF TPA COMPOUNDS. L = NH3 OR PLANAR AMINE, L' = PLANAR

AMINE, X = CL, CARBOXYLATE...... 20

viii

FIGURE 2.2. INFLUENCE OF SERUM BINDING ON PLATINUM CELLULAR UPTAKE IN A2780 OVARIAN

CARCINOMA. A2780 CELLS WERE TREATED WITH 10 µM CISPLATIN AND TPA COMPOUNDS

BEFORE AND AFTER PRETREATMENT WITH SERUM. FROM REF (63)...... 22

FIGURE 2.3. SYNTHETIC SCHEME OF TPA COMPOUNDS. L= 4-PICOLINE, ISOQUINOLINE; R=H

(FORMATE), R=CH3 (ACETATE), R=CH2OH (HYDROXYACETATE), R=CHCH3OH

(LACTATE)...... 23

FIGURE 2.4. STRUCTURE OF TRANS-PLATINUM PLANAR AMINE (TPA) COMPOUNDS SYNTHESIZED. 25

FIGURE 2.5. STRUCTURES OF TPA COMPOUNDS STUDIED...... 27

FIGURE 2.6. STRUCTURES OF N-ACETYL-METHIONINE...... 30

FIGURE 2.7. SUBSTITUTION REACTION OF TPA3 WITH N-ACETYL-METHIONINE (1:2) FOLLOWED BY

1H NMR SPECTROSCOPY. A. SHIFT OF THE H2 OF ISOQUINOLINE. B. FORMATION OF FREE

CARBOXYLATE...... 30

FIGURE 2.8. STRUCTURE OF ETHIDIUM BROMIDE (ETBR) ...... 32

FIGURE 2.9. DEPENDENCIES OF (A). ETHIDIUM BROMIDE FLUORESCENCE AND (B). MELTING

TEMPERATURE ON RB (DRUG TO NUCLEOTIDE RATIO) FOR DNA MODIFIED BY VARIOUS

PLATINUM COMPLEXES IN 10 MM NACLO4 AT 37 °C FOR 48 H. (X) CISPLATIN; (●)

TRANSPLATIN; (∆) [PTCL(DIEN)]CL; (○) TRANS-[PTCL2(NH3)(QUIN)]. FROM REF (124). .... 33

FIGURE 2.10. QUENCHING OF ETHIDIUM BROMIDE FLUORESCENCE INTENSITY UPON TREATMENT

WITH PLATINUM COMPOUNDS AFTER 48 H INCUBATION...... 35

FIGURE 2.11. DNA MELTING TEMPERATURE OF TPAS. 200 ΜM CALF THYMUS DNA SAMPLES

(EXPRESSED AS NUCLEOTIDES) WERE INCUBATED WITH THE PLATINUM COMPLEXES AT

VARIOUS IN 10 MM NACLO4 AT 37 °C FOR 24 H...... 35

FIGURE 3.1. MTT ASSAY TO MEASURE CELL SURVIVAL AND PROLIFERATION...... 38

FIGURE 3.2. CYTOTOXICITY CURVE OF TPA COMPOUNDS AND CISPLATIN BEFORE AND AFTER

INCUBATION WITH SERUM IN A2780 CELLS. PLATINUM COMPOUNDS WERE INCUBATED WITH

FETAL BOVINE SERUM FOR 24 H AND 48 H THEN TREATED CELLS FOR 72 H. GROWTH

INHIBITION WAS THEN MEASURED BY MTT AFTER DRUG TREATMENT...... 41

FIGURE 3.3. STRUCTURES OF TPA COMPOUNDS STUDIED...... 42

FIGURE 3.4. CELLULAR ACCUMULATION OF PLATINUM COMPOUNDS IN A2780 CELLS. CELLS WERE

TREATED FOR 12 H WITH 10 µM OF PLATINUM COMPOUNDS FOLLOWING INCUBATION WITH OR

WITHOUT SERUM FOR 48 H. AFTER THE TREATMENT WITH EACH COMPOUND, CELLS WERE

HARVESTED AND PLATINUM UPTAKE WAS MEASURED ON VARIAN ICP-MS. ERROR BARS

INDICATE MEAN ± S.E.M. FROM TWO INDEPENDENT EXPERIMENTS...... 43

FIGURE 3.5. EFFECTS OF TREATMENT WITH PLATINUM COMPOUNDS ON CELL CYCLE. A2780 CELLS

WERE TREATED WITH 10 µM OF EACH PLATINUM COMPOUNDS FOR 48 H. CELLS WERE THEN

HARVESTED, STAINED WITH PROPIDIUM IODIDE, AND THEN ANALYZED USING FLOW

CYTOMETRY...... 45

FIGURE 3.6. BIFUNCTIONAL DNA CROSSLINKS AND DNA-PROTEIN CROSS-LINKS INDUCED BY TPA.

FROM REF (77)...... 46

FIGURE 4.1. BBR3464 AND ITS NONCOVALENTLY BINDING ANALOGUES, AH44 AND TRIPLATIN NC

...... 53

FIGURE 4.2. CELLULAR ACCUMULATION OF BBR3464, AH44 (I), AND TRIPLATIN NC (II). FROM

REF (103)...... 54

FIGURE 4.3. INHIBITION OF HEPARINASE I BY CISPLATIN, PPC, AND R9. FROM REF (155)...... 55

FIGURE 4.4. PROPOSED TARGET STRUCTURES OF PPC-NC (WITH DANGLING AMINE) AND AH44

(WITH NH3)...... 56

FIGURE 4.5. 1H NMR SPECTRA OF DIFFERENT BBR3464 SYNTHETIC METHODS. A. 1H NMR

SPECTRUM OF IMPURE BBR3464 RESULTED FROM PREVIOUSLY PUBLISHED METHOD

(162,163). B. 1H NMR SPECTRUM OF PURE BBR3464 OBTAINED WITH NEW SYNTHETIC

APPROACH...... 57

FIGURE 4.6. A COMPARISON OF 1) PREVIOUS APPROACH FROM REF (103) AND 2) NEW SYNTHETIC

APPROACH TO SYNTHESIZE TRIPLATIN NC...... 58

FIGURE 4.7. NEW SYNTHETIC SCHEME OF PPC-NC. A. 2 EQ. AGNO3 THEN 2.2 EQ. N-BOC-1,6-

HEXANEDIAMINE IN DMF, B. 0.1M HNO3. C. 1 EQ. AGNO3 THEN 0.5 EQ HEXANEDIAMINE IN

DMF. D. 2.2 EQ. DIPEA E. MONO-ACTIVATED TRANSPLATIN, T-[PT(NH3)2(CL)(NO3)]...... 59

FIGURE 4.8. STRUCTURE OF SYNTHESIZED PPC-CL...... 60

FIGURE 4.9. 1H NMR SPECTRA OF A) MONOPLATIN NC, B) DIPLATIN NC, C) TRIPLATIN NC, D)

TETRAPLATIN NC. A, C, D WERE OBTAINED BY 300MHZ NMR WHILE B WAS OBTAINED BY

600MHZ NMR...... 65

FIGURE 5.1. STRUCTURE OF PPC-NC STUDIED IN THIS CHAPTER...... 68

FIGURE 5.2. HYDROGEN BONDS BETWEEN TRIPLATIN NC AND DNA. FROM REF (98). (A) VIEW

PERPENDICULAR TO THE HELICAL AXIS. (B) VIEW ALONG THE HELICAL AXIS. ATOMS ARE

COLORED BY TYPE WITH CARBON (DNA), GREEN; CARBON (TRIPLATIN NC) GRAY; NITROGEN,

BLUE; OXYGEN, RED; PLATINUM, YELLOW...... 69

FIGURE 5.3. ARGININE FORK (LEFT) AND PHOSPHATE CLAMP (RIGHT)...... 70

FIGURE 5.4. PICTORIAL REPRESENTATION SHOWING THE MECHANISM OF DNA CONDENSATION BY

CATIONIC MOLECULES. MODIFIED FROM REF (170)...... 71

FIGURE 5.5. REPRESENTATIVE AFM IMAGES OF LINEARIZED NEGATIVELY SUPERCOILED PLASMID

DNA, PSP73, IN THE PRESENCE OF PPC-NC. FROM REF (177)...... 72

FIGURE 5.6. ITC PROFILE FOR THE BINDING OF TRIPLATIN NC WITH CT-DNA. THE ISOTHERM IS

DIVIDED INTO 3 STAGES: 1) 1ST BINDING STAGE 2) CONDENSATION 3) PRECIPITATION...... 76

FIGURE 5.7. ITC PROFILES FOR THE BINDING OF PPC-NC TO CT-DNA. A) MONOPLATIN NC(4+),

B) DIPLATIN NC(6+), C) TRIPLATIN NC(8+), D) TETRAPLATIN NC(10+), E) AH44(6+). .... 76

FIGURE 5.8. CD SPECTRA OF 3 MM OF CT-DNA AT VARIOUS CONCENTRATION OF

HEXAMMINECOBALT (III). (1) RI = 0; (2) RI =0.098;(3) RI =0.197; (4) RI =0.216; (5) RI =0.256;

(6) RI =0.275; (7) RI =0.295; (8) RI =0.314; (9) RI =0.334; (10) RI =0.353. FROM REF (189). 79

FIGURE 5.9. CD SPECTRA OF CT-DNA TREATED WITH VARIOUS AMOUNTS OF PLATINUM

COMPOUNDS. 100 µM OF CT-DNA PREPARED IN 10 MM NACLO4 WERE TREATED WITH

PLATINUM COMPOUNDS AT DIFFERENT RI (DNA TO DRUG RATIO) AND INCUBATED FOR 1 H. 80

FIGURE 5.10. CHANGES OF THE ELLIPTICITY AT 210 NM (TOP) AND 274 NM (BOTTOM) UPON

TREATMENT WITH DIFFERENT RI (DNA TO PLATINUM COMPOUNDS)...... 81

FIGURE 5.11. COMPARISON OF THE COMPOUNDS AT RI= 0.05 (TOP) AND RI=0.1 (BOTTOM)...... 82

FIGURE 5.12. STRUCTURES OF DIPLATIN NC, AH44, AND TRIPLATIN NC ...... 83

FIGURE 5.13. CELLULAR ACCUMULATION (ATTOMOLE OF PT/CELL) OF PPC-NC AND AH44 IN

HCT116 CELLS...... 86

FIGURE 5.14. CELLULAR ACCUMULATION (ATTOMOLE OF DRUG/CELL) OF PPC-NC AND AH44 IN

HCT116 CELLS...... 87

FIGURE 5.15. CYTOTOXICITY OF PPC-NC DETERMINED BY MTT ASSAY...... 88

FIGURE 5.16. PATHWAY FOR THE CATIONIC PTDS (R9) INTO CELLS. FROM REF (192) ...... 90

xii

FIGURE 5.17. MAJOR AND MINOR REPEATING DISACCHARIDE SEQUENCES OF HEPARIN. FROM REF

(193)...... 91

FIGURE 5.18. GAG DEPENDENT CELLULAR ACCUMULATION OF PPC-NC. 2 X 106 CELLS WERE

TREATED WITH 10 µM PLATINUM COMPOUNDS. AFTER 1 H, CELLS WERE HARVESTED AND

WASHED WITH PBS BUFFER. AFTER ACID DIGESTION, PLATINUM ANALYSIS WAS PERFORMED

ON ICP-OES. FROM REF (156)...... 92

FIGURE 5.19. ARGININE FORK, PHOSPHATE CLAMP, AND SULFATE CLAMP...... 93

FIGURE 5.20. CELLULAR ACCUMULATION OF PPC-NC COMPOUNDS IN CHO WT AND CHO-745

HSPG DEFICIENT CELLS. CELLS WERE TREATED WITH 10 µM OF PLATINUM COMPOUNDS FOR 3

H...... 94

FIGURE 5.21. STRUCTURE OF 15N LABELLED BBR3464...... 95

FIGURE 5.22. STRUCTURE OF GLUCOSAMINE-3-SULFATE...... 96

FIGURE 5.23. HSQC [1H, 15N] OF 2 MM OF BBR3464 WITH 8 MM GLUCOSAMINE-3-SULFATE

(G3S). A) BBR3464 WITHOUT G3S. B) BBR3464 WITH G3S AFTER 72 H. NH3 REGION: (I)

PTN3CL, (II) PTN4. NH2 REGION: (III) PTN4. PTN3CL IN NH2 REGION IS NOT SHOWN IN THIS

SPECTRA SINCE IT OVERLAPS WITH THE WATER PEAK...... 97

FIGURE 5.24. ISOTHERMAL TITRATION CALORIMETRY OF BBR3464 AND G3S...... 98

FIGURE 5.25. STRUCTURE OF METHYLENE BLUE (MB) DYE...... 98

FIGURE 5.26. ABSORPTION SPECTRA OF STANDARDS. TO 150 µL OF HEPARIN SAMPLES AT VARIOUS

CONCENTRATIONS (0 µG/ML TO 10 µG/ML), 150 µL OF MB SOLUTION WAS ADDED, THEN

MEASURED IMMEDIATELY...... 100

FIGURE 5.27. CALIBRATION CURVE AT 650 NM...... 101

xiii

FIGURE 5.28. FULL ABSORPTION SPECTRA UPON TREATMENT WITH PPC-NC. 150 µL OF 10 µG/ML

OF HEPARIN SULFATE SOLUTION WAS TREATED WITH 2.4 µM OF PPC-NC. TO THIS MIXTURE,

150 µL OF MB SOLUTION WAS ADDED THEN SUBSEQUENTLY MEASURED BY UV/VIS

SPECTROSCOPY...... 102

FIGURE 5.29. ABSORBANCE INTENSITY AT 650 NM AFTER TREATMENT WITH PLATINUM

COMPOUNDS. 150 µL OF 10 µG/ML OF HEPARIN SULFATE SOLUTION WAS TREATED WITH 2.4

µM OF PPC-NC. TO THIS MIXTURE, 150 µL OF MB SOLUTION WAS ADDED THEN

SUBSEQUENTLY MEASURED BY UV/VIS SPECTROSCOPY...... 102

FIGURE 5.30. EFFECTS OF HEPARIN SULFATE ON DNA BINDING. A) 100 µM OF CT-DNA WAS

INCUBATED WITH 30 µG/ML HEPARIN SULFATE FOR 1 H. B) 100 µM OF CT-DNA WAS

INCUBATED WITH 10 µM OF TRIPLATIN NC FOR 1 H. C) 10 µM OF TRIPLATIN NC WAS

INCUBATED WITH 60 µG/ML HEPARIN SULFATE FOR 1 H, THEN 100 µM OF CT-DNA WAS

ADDED AND INCUBATED FOR ADDITIONAL 1 H. D) 100 µM OF CT-DNA WAS INCUBATED WITH

10 µM OF TRIPLATIN NC FOR 1 H, THEN 60 µG/ML HEPARIN SULFATE WAS ADDED AND

INCUBATED FOR ADDITIONAL 1 H...... 104

FIGURE 5.32. EFFECTS OF HEPARINASE I ON PPC NC AND HEPARIN ADDUCT. A) 100 µM OF CT-

DNA B) 10 U/ML OF HEPARINASE I C) 100 µM OF CT-DNA WAS INCUBATED WITH 10 µM OF

TRIPLATIN NC FOR 1 H, THEN 60 µG/ML HEPARIN SULFATE WAS ADDED AND INCUBATED FOR

ADDITIONAL 1 H. C) 60 µG/ML HEPARIN SULFATE WAS INCUBATED WITH 10 µM OF TRIPLATIN

NC FOR 1 H, THEN 100 µM OF CT-DNA WAS ADDED AND INCUBATED FOR ADDITIONAL 1 H.

D) 30 µG/ML HEPARIN SULFATE WAS INCUBATED WITH 10 µM OF TRIPLATIN NC FOR 1 H,

THEN 100 µM OF CT-DNA WAS ADDED AND INCUBATED FOR ADDITIONAL 1 H. TO THE

MIXTURE, HEPARINASE I WAS ADDED AND INCUBATED FOR 0.5 H...... 105

xiv

FIGURE 6.1. METABOLITES OF PPC-CL ANALOGS PRODUCED UPON METABOLIC DEGRADATION BY

SULFUR NUCLEOPHILES. TRI-, TETRA-, AND PENTAPLATIN-CL RELEASES MONO-, DI-, AND

TRIPLATIN NC, RESPECTIVELY...... 110

FIGURE 6.2. SYNTHETIC SCHEME OF PENTAPLATIN-CL ...... 111

FIGURE 6.3. PROPOSED STRUCTURES OF PPC-CL...... 113

FIGURE 6.4. INACTIVE METABOLITES PRODUCED BY PPC UPON THE INTERACTION WITH NAC. .. 113

FIGURE 6.5. REACTION OF PPC WITH NAC FOLLOWED BY ESI-MS. I. BBR3464 WITH NAC. II.

TETRAPLATIN-CLWITH NAC. III. PENTAPLATIN-CL WITH NAC. A. PPC-NC: MONOPLATIN

NC(I), DIPLATIN NC(II), AND TRIPLATIN NC (III). B. INACTIVE METABOLITE:

+ [PT(NH3)2(NAC)2] . C. THIOLATE BRIDGED INACTIVE METABOLITE [(NAC)(NH3)2PT-NAC-

+ PT(NAC)(NH3)2] . (THE STRUCTURES OF B AND C ARE SHOWN IN FIGURE 6.4)...... 114

FIGURE 6.6. REACTION OF PPC-CL WITH NAC FOLLOWED BY 195PT NMR SPECTROSCOPY. A.

TETRAPLATIN-CL, B. TETRAPLATIN-CL WITH NAC...... 115

FIGURE 6.7. SYNTHETIC SCHEME OF BBR3464-ACETATE...... 117

FIGURE 6.8. STRUCTURE OF RGD TRIPEPTIDE ...... 119

FIGURE 6.9. STRUCTURE OF TRANS-PT[(ISQ)(NH3)(SUCCINATE)2]...... 119

FIGURE 6.10. ATTEMPT TO SYNTHESIZE TRANS-PT[(ISQ)(NH3)(SUCCINATE)2] BY USING

METHYLATED SUCCINATE ...... 120

FIGURE 6.11. SYNTHESIS OF TRANS-PT[(ISQ)(NH3)(SUCCINATE)2] BY USING EXCESS SUCCINIC ACID.

...... 122

FIGURE 6.12. CONJUGATION OF RGD PEPTIDE TO TPA...... 123

FIGURE 6.13. PROPOSED STRUCTURES OF PPC-NBD COMPOUNDS ...... 124

FIGURE 6.14. PREPARATION OF 6-NBD LIGAND...... 125

FIGURE 6.15. GENERAL SCHEME OF NBD-CONJUGATION ...... 126

FIGURE 6.16. 1H NMR SPECTRA OF 6-NBD LIGAND (TOP) AND MONOPLATIN-NBD (BOTTOM).

DUE TO DIFFERENCE IN SOLUBILITY, 1H NMR SPECTRUM OF 6-NBD WAS OBTAINED IN

CD3OH WHILE THE SPECTRUM OF MONOPLATIN-NBD WAS OBTAINED IN D2O...... 128

FIGURE 6.17. PROPOSED SYNTHETIC SCHEME OF FLUORINE-18 LABELLED CISPLATIN ...... 130

FIGURE 6.18. RECOVERY OF K2PTCL4 FROM PLATINUM WASTE...... 132

xvi

LIST OF TABLES

TABLE 1.1. SUMMARY OF CISPLATIN RESISTANCE MECHANISM AND THE CIRCUMVENTION OF THE

RESISTANCE WITH TPA CARBOXYLATES COMPOUNDS...... 10

TABLE 1.2. SUMMARY AND COMPARISON OF STRUCTURAL CHARACTERISTICS OF DNA CROSS-

LINKS OF TRANS-[PTCL2(NH3)(THIAZOLE)], TRANSPLATIN, AND CISPLATIN. FROM REF (50).

...... 12

TABLE 1.3. SUMMARY AND COMPARISON OF STRUCTURAL CHARACTERISTICS OF PT-DNA

ADDUCTS OF DIFFERENT CLASSES OF PLATINUM COMPOUNDS...... 15

TABLE 2.1. IC50 VALUES OF PLATINUM COMPOUNDS IN HCT116 HUMAN COLON CARCINOMA.

FROM REF (71)...... 21

TABLE 2.2. INITIAL HYDROLYSIS RATE, AQUEOUS SOLUBILITY, AND METHIONINE BINDING RATE OF

TPA COMPOUNDS. VALUES IN PARENTHESES ARE FROM REF (66), WHERE THE VALUES WERE

OBTAINED IN PBS BUFFER...... 29

TABLE 3.1. CYTOTOXICITY OF TPA COMPOUNDS AND CISPLATIN BEFORE AND AFTER INCUBATION

WITH SERUM IN A2780 CELLS...... 41

TABLE 4.1. CYTOTOXICITY OF CISPLATIN (C-DDP), BBR3464, AH44 (I), AND TRIPLATIN NC (II)

IN VARIOUS TUMOR CELLS. FROM REF (103)...... 54

TABLE 5.1. BEST FIT PARAMETERS FOR TITRATION OF CT-DNA WITH PLATINUM COMPOUNDS.

THESE VALUES WERE OBTAINED BY PLOTTING THE BEST FIT ON THE INITIAL BINDING STAGE OF

THE COMPOUNDS WITH CT-DNA BY USING ORIGIN 7.0. A) FROM REF (181). N.R= NOT

REPORTED...... 77

TABLE 5.2. CYTOTOXICITY OF PPC- NC COMPOUNDS DETERMINED BY MTT ASSAY IN HCT116

CELLS...... 88

xvii

List of Abbreviations

4-pic 4-picoline (4-methylpyridine)

A2780 human ovarian carcinoma

AFM atomic force microscopy boc tert-butoxy carbonyl

CD circular dichroism

CHO chinese hamster ovary cisplatin cis-diamminedichloroplatinum(II)

CS chondroitin sulfate

CT calf thymus

CTR1 copper transporter protein

D2O deuterium oxide

DACH diaminocyclohexane

DIPEA diisopropylethylamine

DMF dimethylformamide

DNA deoxyribose nucleic acid

ESI electrospray ionization

EtBr ethidium bromide

FBS Fetal bovine albumin

FDA food and drug administration form formate

GAG glycosaminoglycans

GHP general purpose hydrophilic polypropylene

xviii

GSH glutathione

HCT116 human colon carcinoma

HMG high mobility group protein

HPLC high performance liquid chromatography

HSPG heparan sulfate proteoglycan

HS heparin sulfate

HSA human serum albumin

HSPG heparan sulfate proteoglycan

HSQC heteronuclear single quantum coherence

IC50 half maximal inhibitory concentration

ICP inductively coupled plasma isq isoquinoline

ITC tsothermal titration calorimetry lact lactate m/z mass to charge ratio

MB methylene blue

MS mass spectrometry

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide

NAC n-acetyl-methionine

NAM n-acetyl-cysteine

NBD 7-nitro-2,1,3-benzoxadiazole

NC noncovalent

NER nucleotide excision repair

xix

NMR nuclear magnetic resonance

OAc acetate

OAcOH hydroxyacetate

PBS phosphate buffer saline

PET positron emission tomography

Pic picoline

PPC polynuclear platinum compounds

PPC-Cl polynuclear platinum compounds chloride analogs

PPC-NC noncovalently binding polynuclear platinum compounds

[PtCl(dien)]Cl chlorodiethylenetriamineplatinum(II) chloride

Pt platinum

PTDs Protein transduction domains

Py pyridine

Quin quinolone

RGD Arginylglycylaspartic acid

Ri drug to nucleotide ratio

TCAP K[PtCl3(NH3)]

TPA trans-Platinum Planar Amine

Tz thiazole

UV ultraviolet

Vis visible v:v volume to volume wt wild type

ABSTRACT

DEVELOPMENT OF NON-TRADITIONAL PLATINUM ANTICANCER AGENTS: TRANS- PLATINUM PLANAR AMINE COMPOUNDS AND POLYNUCLEAR PLATINUM COMPOUNDS

By Daniel E. Lee, Ph.D.

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University.

Virginia Commonwealth University, 2015

Major Director: Nicholas P Farrell, Ph.D., Professor, Department of Chemistry

Platinum anticancer compounds with cis geometry, similar to cisplatin, have been explored to circumvent the cisplatin resistance; however, they were not considered broadly active in cisplatin cells due to exhibiting similar or same cell death mechanism as cisplatin. Platinum compounds with trans geometry were less studied due to transplatin being clinically inactive; but with few structural modifications, they resulted in unaffected cytotoxic activities in cisplatin resistant cells with structural modification by exhibiting different modes of DNA binding. This research focused on further exploring and establishing structure-activity relationship of two promising non-classical series of platinum compounds with trans-geometry: trans-platinum planar amine (TPA) compounds and noncovalently binding polynuclear platinum compounds (PPC-NC).

During this research, further optimizations of the reactivity of TPA compounds were accomplished by modifying the leaving carboxylate groups. The effects of modified reactivity were probed by a systematic combination of chemical and biophysical assays, followed by

xxi evaluating their biological effects in cells. To establish the structural-activity relationship of PPC-

NCs, Mono-, Di-, Tri-, and Tetraplatin NC with charge of 4+, 6+, 8+, and 10+ were synthesized and evaluated by utilizing biophysical and biological assays. Lastly, a new class of polynuclear platinum compounds, Hybrid-PPCs, were synthesized and evaluated to overcome the pharmacokinetic problems of BBR3464, phase II clinical trial anticancer drug developed previously in our laboratory.

xxii

Chapter 1

Introduction

CHAPTER 1. INTRODUCTION

1.1 Cancer

Cancer is a group of diseases characterized by the uncontrolled growth and spread of abnormal cells with a specific capacity to invade and destroy tissues and organs almost anywhere in the body. Although the causes of cancer are not apparent, 90-95% of cancers are thought to be caused by environmental factors: tobacco, diet and obesity, infections, radiation, stress, lack of physical activity, and environmental pollutants (1). Approximately 5–10% of cancers are thought to be caused due to genetic defects inherited from a person's parents (1). While the majority of the cancers are preventable, continuous research efforts should be made to cure cancer since it is the second most common cause of death in the United States, accounting for nearly 1 of every 4 deaths

(2).

Figure 1.1. The role of genes and environment in the development of cancer. A. The percentage contribution of genetic and environmental factors to cancer. B. Family risk ratios for selected cancers. C. Percentage contribution of each environmental factor From ref. (1).

1.2 Treatments

Current treatment of cancer includes surgery, chemotherapy, radiation therapy, hormonal therapy, targeted therapy, and palliative care. Treatments used depend upon the type, location, and grade of the cancer as well as the person’s health and desires (3). Improvements in curability and survivability are dependent on advances in early detection, surgery, radiotherapy, and chemotherapy; but once widespread metastatic disease has become established, chemotherapy is a central component of management (4). Treatments like radiation and surgery are considered as local treatments since they act only in one area of the body. Chemotherapy differs from surgery or radiation in that it is almost always used as a systemic treatment.

The era of chemotherapy began in the 1940s with the first uses of nitrogen mustards with antifolate drugs (5). Since then, many categories of chemotherapy drugs have been developed as a single agent or combination therapy: alkylating agents, antimetabolites, anti-tumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, and corticosteroids (3). Despite significant advances in chemotherapy, most of the cancer drugs have shown to induce toxic side effects and to develop drug resistances (6).

1.3 Platinum-Based Chemotherapeutics

Cisplatin (cis-diamminedichloroplatinum(II)) was first described in 1845 and its antitumor properties were accidently found by Rosenberg in 1965 (7,8). Since then platinum based antitumor agents have been commonly used for the treatment of a wide range of solid tumors. Structurally, cisplatin or cis-[Pt(NH3)2Cl2], consists of two inert ammine ligands and two labile chloride ligands in cis conformation as shown in Figure 1.2. Platinum (II), exhibiting square planar geometry, tends

3 to have a high affinity for nitrogen and sulfur molecules found commonly in proteins and nucleobases in DNA

Figure 1.2. FDA approved platinum-based anticancer agents

The U.S. Food and Drug Administration (FDA) has approved three platinum based antitumor agents, cisplatin and its derivatives, carboplatin and oxaliplatin, as single agents or as part of combinatorial chemotherapy. Despite the clinical success of cisplatin and its derivatives, dose-limiting toxic side effects and inherent and acquired resistance against approved platinum compounds prompted the development of a new generation of antitumor agents (9,10,11).

1.4 Mechanism of Action of Cisplatin

Following administration of cisplatin into the bloodstream of a patient, replacement of its chlorides by aquation is limited due to high chloride concentration in the blood plasma (~100 mM).

Once it enters the cell through passive diffusion or by active transporter (e.g. copper transporter

Ctr1) (12,13), it is more vulnerable to aquation due to much lower chloride concentration (~4 mM) than the plasma (14). In cells, the drugs are likely to undergo hydrolysis to form positively charged mono- and di-aquated active species by replacement of the chloride leaving group, resulting in subsequent interactions with cellular nucleophiles (Figure 1.3) (15).

Figure 1.3. Mechanism of action of cisplatin

The ultimate target for cisplatin inside the cell is DNA and the active (aquated) species of cisplatin can readily react with preferentially G7 on DNA bases guanine and adenine (16,17).

Initially, mono-functional adducts are formed, but most of them react further to form bi-functional intra- and inter-strand cross-links, which induce structural distortions on DNA (Figure 1.4) (11,18).

Most adducts occur in the same DNA strand and involve adjacent bases, namely GpG 1,2 intrastrand (60-65% of all adducts) and ApG 1,2 intrastrand cross-links (20-25%) (19,20). These cross-links bend DNA helix by 34° towards its major groove and unwind it by 13° (21); these

5 structural distortions can impede cellular processes such as replication and transcription, ultimately leading to apoptosis (22).

Figure 1.4. Formation of cisplatin adducts. From ref (23).

1.5 Cisplatin Resistance

The occurrence of resistance is a common drawback in cancer chemotherapy, and cisplatin is no exception. Tumors are sensitive to cisplatin treatment predominantly; however, most of the sensitive tumors develop resistance after the initial treatment (24,25). Resistance to cisplatin is generally multifactorial and the principal mechanisms responsible for inherent and acquired resistance are inactivation by sulfur containing molecules, reduced cellular accumulation due to change of the uptake/efflux profile, and increased recognition and repair of cisplatin-DNA adducts

(11,26,27).

Although DNA is the core target for the vast majority of platinum anti-cancer compounds, the critical step in measuring the clinical effectiveness of these drugs is to understand and measure the metabolic interactions that may occur while traveling to the desired site of action. Platinum in vivo, following aquation, acts as soft Lewis acids, and form stable complexes with S or N donors found in proteins (28). Reaction with human serum albumin (HSA), a single chain protein

6 containing 17 disulfide bridges and one free thiol at Cys-34, is thought to be the main route for inactivation in human blood plasma (29). Another known biomolecule responsible for cisplatin inactivation is glutathione (GSH), an abundant thiol-containing tripeptide, which is present in a concentration of 1~10 mM intracellularly (30). Clinical and preclinical studies of cellular systems with elevated levels of HSA and GSH are significantly more resistant to cisplatin (31). From these findings, therapeutic efficacy of platinum compound can be reflected as a balance between metabolic inactivating interactions and biological consequence of DNA toxicity.

Figure 1.5.Structure of sulfur nucleophiles: Cysteine, methionine, and glutathone.

Widely used second generation platinum-based anticancer drugs, carboplatin and oxaliplatin, were developed to address the pharmacokinetic problems of cisplatin by reducing the reactivity of the drug. This was accomplished by replacing reactive chloride with a more stable bidentate carboxylate leaving group (32). This approach successfully minimized the interaction with sulfur containing molecules, where this interaction is known to be responsible for a variety of biological effects including drug inactivation, development of resistance, and toxic side effects

(33). Although utilizing more stable carboxylates resulted in enhanced stability to sulfur nucleophiles, they are not considered broadly active in cisplatin-resistant cancers since they form similar Pt-DNA adducts at the same sites on DNA as cisplatin (34,35,36,37,38).

Figure 1.6. Resistance to cisplatin and carboplatin mediated after DNA binding. From ref (10).

Formation and persistence of DNA adducts of cisplatin and its derivatives are crucial in inducing apoptosis. Therefore, an increased rate of Pt-DNA adduct repair or removal of these adducts will attenuate the apoptotic process, which has been associated with cisplatin resistance

(39,40,41,42). At present, several mechanisms of tolerance and removal of cisplatin-DNA adducts are identified and shown in Figure 1.6. These cisplatin resistant mechanisms through recognition and repair are generally shared with its cis derivatives since it forms same or similar adducts with

DNA and is recognized by the proteins responsible for the repair mechanisms. Interestingly, this cross-resistance was less for oxaliplatin, which is thought to be a result of additional steric hindrance presented by the diamocyclohexane (DACH) carrier ligand (43,44,45).

Figure 1.7. Structures of trans-PtTz, trans-[PtNH3(thiazole)Cl2], and BBR3464, [{trans-PtCl(NH3)2}2(µ-

4+ trans-Pt(NH3)2{NH2(CH2)6NH2}2)] In contrast, structurally unique trans-platinum planar amine (TPA) and polynuclear platinum compounds (PPC), resulted in unaltered cytotoxic activities in both cisplatin and oxaliplatin-resistant cells (46,47,48,49). This was exemplified with trans-PtTz and BBR3464

(Figure 1.7), which were demonstrated to bypass cisplatin recognition and repair mechanisms to cause unaffected cytotoxic activities in resistant cells by binding to DNA in distinct manners

(50,51). Therefore, to develop a compound with a superior or wider spectrum of antitumor efficacy, a compound needs to be designed to interact with DNA in a manner different than cisplatin and its analogues (52).

1.6 Nontraditional Platinum Compounds

1.6.1 trans-Platinum Planar Amine (TPA) Compounds

Figure 1.8. Structure of cisplatin and transplatin

After the discovery of cisplatin, the cis configuration of Pt(II) compounds was extensively studied, since early structure-activity studies showed that a cis configuration of the two leaving

9 groups is essential for antitumor activity of simple Pt(II) derivatives. This has also been confirmed for several groups of compounds (7,8) and the trans configuration was believed to be clinically inactive (8,53,54,55,56). It is generally accepted that the trans isomer of cisplatin is inactive due to two major factors: kinetic instability allowing faster deactivation and lack in its ability to induce

DNA structural perturbation. However, several classes of trans-Pt compounds violated this rule and exhibited remarkable cytotoxic activities by interacting with DNA in a unique manner.

Furthermore, they were active in tumor cells resistant to cis analogues (57,58,59). Among such complexes are those with bulky N-donor aromatic heterocycles, iminoethers, cyclohexylamine, and aliphatic amines (60). These cytotoxic trans platinum complexes induce qualitatively and quantitatively different DNA adducts, indicating that cisplatin-like DNA adducts are not mandatory determinants for antitumor activity of platinum compounds (61).

Table 1.1. Summary of cisplatin resistance mechanism and the circumvention of the resistance with TPA carboxylates compounds.

Molecular Mechanism References Circumvention of the References drug resistance with TPA

Inactivation of cisplatin by (62,63,64) Enhanced stability (65,66) sulfur nucleophiles

Reduced accumulation by (27,67,68,69) Enhanced cellular (50,70,71,58) changing the profile of accumulation influx/efflux

Increased repair/tolerance/ (72,73,74,75,76) Formation of different Pt- (58,77,78,79,80) removal and failure of DNA adducts. Different apoptotic pathways cell death mechanism

As described in the previous section, the major obstacle of current platinum chemotherapy is the occurrence of inherent and acquired resistance of tumor cells, and it is of considerable interest to identify an active compound in the resistant cells. The known mechanisms of cisplatin resistance are shown in Table 1.1 and TPA compounds circumvented these mechanisms by demonstrating higher stability to sulfur nucleophiles, enhanced cellular accumulation, and formation of different

Pt-DNA adduct ultimately leading to distinct cell death (Table 1.1).

The trans-isomer of cisplatin, transplatin (trans-[Pt(NH3)2Cl2]), does not exhibit a comparable chemotherapeutic effect and is considered clinically inactive. When one or both of the

NH3 groups in transplatin are replaced to a sterically hindered planar amine ligand, the compound exhibits micromolar cytotoxicity equivalent to that of cisplatin and retains cytotoxicity toward cisplatin- and oxaliplatin-resistant cell lines. A possible explanation for this retained cytotoxic activity is associated with its distinct DNA binding profile exhibited by the trans-platinum compounds (50,81).

Figure 1.9. Cross-links formed by trans-PtTz. From Ref (82)

Table 1.2. Summary and comparison of structural characteristics of DNA cross-links of trans-

[PtCl2(NH3)(thiazole)], transplatin, and cisplatin. From Ref (50).

The unique DNA binding of TPA was exemplified with trans-PtTz as shown in Figure 1.9 and Table 1.2. trans-PtTz exhibited significantly higher formation of interstrand cross-links with

DNA compared to cisplatin or transplatin (83). The steric effects of these planar ligands resulted in the formation of structurally unique cross-links, which may be the main cause of its unique cellular effects being significantly different from that of cisplatin. Despite high antitumor activity and distinct mode of binding, trans-PtTz is more reactive than the cis-isomer due to high trans- influencing chlorides trans to one another, whereas this increased reactivity potentially increases its susceptibility to inactivation by sulfur nucleophiles. This is likely to have a major influence in the biological activities of TPA and further optimization is made to address this pharmacokinetic problem.

Figure 1.10. Rational design of TPA compounds. 12

To further optimize the pharmacological properties of the TPA compounds, the chloride leaving group can be replaced with carboxylate leaving groups. This greatly enhances aqueous solubility and stability to hydrolysis while retaining the cytotoxicity of the parent chloride compounds (65,70,84,71). The utilization of a carboxylate leaving group was previously exemplified with carboplatin and oxaliplatin by utilizing bi-dentate carboxylate leaving groups to significantly enhance their stability compared to cisplatin. TPA carboxylates utilizes two monodentate carboxylate leaving groups of TPA compounds in trans position to another, which allows enhanced stability due to the weak trans influencing nature of the carboxylate leaving group. This strategy is rather novel and further reactivity optimization of TPA is necessary to identify a clinically relevant chemotherapeutic platinum agent.

1.6.2 Polynuclear Platinum Compounds (PPC)

Figure 1.11. Structure of BBR3464.

In efforts to differentiate the DNA binding of platinum compounds from cisplatin, another innovative approach was achieved by the syntheses of cationic polynuclear platinum complexes

(PPC). These compounds contain two or three platinum centers with both cis and trans configurations connected by polyamine linkers. A representative trinuclear complex, 4+ charged

BBR3464, is the first polynuclear platinum compound to enter clinical trials as an anticancer agent.

BBR3464 showed promising results by demonstrating higher cellular uptake and exhibiting up to hundred fold increase in potency on a molar dosage than cisplatin. BBR3464 also showed unaffected potency in cisplatin-resistant and p53 mutant tumors (85,86,87,88,89,90).

Figure 1.12. Structures from molecular modeling of the major DNA adducts of BBR3464. From Ref (82).

The overall DNA binding profile of BBR3464 is significantly different from that of cisplatin. Due to the positively charged nature of polynuclear platinum compounds, the molecule initially interacts through noncovalent interaction, where this interaction allows the DNA binding to be significantly more rapid than that of cisplatin (91). Then it forms various unique long-range interstrand and intrastrand bifunctional cross-links as shown in Figure 1.12 (51,92). The unique properties of long-range interstrand cross-links of BBR3464 and resulting conformational alterations in DNA showed critical consequences for their antitumor effects. The platinum-DNA adducts resulted from the unique mode of binding are not recognized by high mobility group domain proteins (HMG) and are a poor substrate for nucleotide excision repair (NER). HMG and

NER proteins are known to be responsible for the recognition and repair of cisplatin-DNA adduct and this may explain the unaffected cytotoxic activities seen in cisplatin resistant cells (92).

Although BBR3464 resulted in a non-cross resistant cytotoxicity with cisplatin by inducing a distinct mode of DNA binding, degradation into inactive metabolites by sulfur nucleophile was no exception for BBR3464 as with other covalently binding platinum compounds. Despite its success demonstrated in pre-clinical and Phase I clinical studies, results from Phase II clinical studies revealed a rapid degradation of BBR3464 into inactive metabolites due to higher plasma protein binding in man than in mice. These pharmacokinetic factors have discouraged the use of

BBR3464 in the clinical setting (93,94).

1.6.3 Noncovalently Binding Cationic Polynuclear Platinum Compounds (PPC-NC)

Table 1.3. Summary and comparison of structural characteristics of Pt-DNA adducts of different classes of platinum compounds.

Platinum Examples Predominant Pt-DNA References Compounds Adducts

Cisplatin and its Cisplatin Short-range intrastrand (1,2) (95,96) analogues Carboplatin cross-links Oxaliplatin

TPA Compounds trans-Pt[(Cl2)(NH3)(Tz)], Short-range interstrand (97,98) trans-Pt[(L)(L’)(NH3)(X2) cross-links

Covalently Binding BBR3464 Long-range interstrand (1,4) (51,99,100) Polynuclear Platinum 1,1/t,t cross-links Compounds (PPC)

Noncovalently Triplatin NC Noncovalent interactions (101,102) Binding Polynuclear with negatively charged Platinum Compounds phosphate backbone (PPC-NC)

Traditional platinum chemotherapeutic agents, cisplatin and its analogues, all form predominantly short-range intrastrand crosslinks on DNA to cause severe conformational distortions, which is a critical determinant of the biological consequence of the platinum chemotherapeutic treatment. The structurally distinct trans-isomer, TPA compounds and polynuclear platinum compounds (PPC) form short-range interstrand cross-links and long-range interstand cross-links with DNA, respectively (summarized in Table 1.3). By exhibiting these distinct modes of DNA binding, the cellular resistance to cisplatin involving enhanced DNA recognition and repair was minimized. One drawback of all covalently binding platinum compounds are their susceptibility to the drug inactivation by binding to sulfur nucleophiles.

Figure 1.13. Structures of trinuclear platinum compounds.

While studying the long-range cross-links with BBR3464 in DNA, a pre-association of the central platinum moiety in the minor groove through noncovalent interaction was observed (92).

To solely study the noncovalent interactions of PPC, the reactive chlorides were replaced with inert ammine ligands and later it was discovered that these inert platinum compounds exhibit antitumor properties (103,104). This novel approach, achieved by replacing reactive chloride leaving group of BBR3464 with substitutionally inert ammine ligand (NH3) or “dangling” amine

(NH2(CH2)6NH3) (Figure 1.13) to produce inert polynuclear platinum agents, completely negated the pharmacokinetics problems of covalently binding platinum compounds due to absence of any covalent binding site (105,103).

Noncovalently binding polynuclear platinum compounds (PPC-NC) demonstrated interesting biological properties including in vitro cytotoxicity and high cellular accumulation in various tumors cells including cisplatin-resistant and p53 mutant cells (106,107). Contradicting the previous assumption that the platinum compounds have to be neutral or hydrophobic to enter the cells more efficiently, Triplatin NC (8+) demonstrated much higher cellular accumulation than those of cisplatin and BBR3464 (4+) (54). Interestingly, Triplatin NC (8+) showed higher cytotoxicity and cellular accumulation than its congener AH44 (6+) despite its higher charge, suggesting a trend that higher charged polynuclear platinum compounds generally demonstrate superior anticancer properties (106).

Yet, PPC-NC and their structure-activity relationships (SAR) have not been explored thoroughly. A further investigation is deserved to establish the SAR including the effects of charge, number of platinum centers, and type of inert ligands on antitumor properties to develop clinically relevant noncovalently binding platinum chemotherapeutics. Moreover, their drug mechanism including the overall profile of DNA interaction, mechanism of cellular internalization, and mechanism of cell death should be investigated further.

1.7 Aim and Scope of This Thesis

Platinum anticancer compounds with cis geometry, similar to cisplatin, have been explored to circumvent the cisplatin resistance; however, they were not considered broadly active in cisplatin cells due to exhibiting similar or same cell death mechanism as cisplatin. Platinum compounds with trans geometry were less studied due to transplatin being clinically inactive; but with few structural modifications, they resulted in unaffected cytotoxic activities in cisplatin resistant cells with structural modification by exhibiting different modes of DNA binding. This thesis focuses on further exploring and establishing structure-activity relationship of two promising non-classical series of platinum compounds with trans-geometry: trans-platinum planar amine (TPA) compounds and noncovalently binding polynuclear platinum compounds (PPC-NC).

In Chapter 2, further optimizations of the reactivity of TPA compounds are accomplished by modifying the leaving carboxylate groups. The effects of modified reactivity are probed by a systematic combination of chemical and biophysical assays, followed by evaluating their biological effects in cells (Chapter 3). Mono-, Di-, Tri-, and Tetraplatin NC with charge of 4+,

6+, 8+, and 10+ are synthesized in Chapter 4 to establish the structural-activity relationship of

PPC-NC by expanding the series. These compounds are evaluated by observing their overall DNA binding profiles, cellular accumulation, and cytotoxic activities (Chapter 5). Other works accomplished throughout the years in the graduate program are reported in Chapter 6.

Chapter 2

Synthesis, Characterization, and Chemical Property of TPA

Compounds

CHAPTER 2. SYNTHESIS, CHARACTERIZATION, AND

CHEMICAL PROPERTY OF TRANS-PLATINUM PLANAR AMINE

COMPOUNDS

Figure 2.1. General structure of TPA compounds. L = NH3 or planar amine, L' = planar amine, X = Cl, carboxylate.

trans-Platinum planar amine compound (TPA) is considered as a strong candidate to circumvent cisplatin resistance by demonstrating enhanced stability and forming distinct Pt-DNA adducts. Further investigations on structural-activity relationships are necessary to ultimately develop a clinically useful drug. The general structure of TPA compounds is trans-Pt[X2(L)(L’)], where L and L’ are the carrier group (L= NH3, planar amine, L’= planar amine and X being the leaving group (chloride, carboxylates) (Figure 2.1). Based on previous observations, the reactivity of the TPA compounds can be modulated by modifying the carboxylate leaving group; and the formation of Pt-DNA adduct can be controlled by utilizing different planar amine carrier ligands

(thiazole, pyridine, picoline, isoquinoline, etc.). This “fine-tuning” strategy of TPA is further explored in this chapter to identify a compound with superior therapeutic efficacy, where a balance between cytotoxic activities and inactivating interactions is accomplished.

2.1 Structure-Based Rational TPA Drug Design

2.1.1 Effects of Planar Amines Carrier Ligands on Cytotoxicity

Table 2.1. IC50 values of platinum compounds in HCT116 human colon carcinoma. From Ref (71).

A previous study (Table 2.1) shows that trans-[Pt(O2CH2)2(NH3)(Isq)] exhibited higher cytotoxicity while the TPA compounds with 4-picoline carrier ligand required higher dosage to cause equivalent amounts of cell death (71). The result suggests that the choice of planar amine carrier ligand is critical in terms of cytotoxicity, where larger and greater steric hindrance isoquinoline ligand resulted in higher cytotoxicity compare to picoline or pyridine compounds.

This trend was also confirmed with other studies, where the order of cytotoxicity exhibited for different carrier ligands with acetate leaving group were found to be as following (highest to lowest): Isoquinoline > 2-picoline > 4-picoline > pyridine (71,84). These results suggest to utilize isoquinoline as a carrier ligand for further developments and investigations.

2.1.2 Effects of Carboxylate Leaving Groups on Cytotoxicity and Cellular

Accumulation

Figure 2.2. Influence of serum binding on platinum cellular uptake in A2780 ovarian carcinoma. A2780 cells were treated with 10 µM cisplatin and TPA compounds before and after pretreatment with serum. From Ref (63).

Previously, it was shown that the nature of the carboxylate affects the cytotoxicity even in closely related compounds. These results are shown in Table 2.1, where the compound with more reactive formate has shown significantly higher cytotoxicity than that with more stable acetate.

Figure 2.2 shows that carboxylate leaving group also affects cellular accumulation and metabolic stability of the TPA compounds. Figure 2.2 shows the cellular accumulation of TPA compounds measured in A2780 ovarian carcinoma before and after serum incubation. Serum is a liquid portion of blood, where most of the inactivation of the platinum compounds occurs due to presence of sulfur nucleophiles (e.g. serum albumin). Although all of the TPA with carboxylates were shown to be significantly more stable than cisplatin, TPA compounds with higher reactivity (formate) resulted in higher cellular accumulation while demonstrating less stability to the protein binding.

From these findings, it is suggested that the reactivity of carboxylate leaving group on TPA contributes crucially to the cytotoxicity, cellular accumulation, and metabolic stability. These results convey the need to further modulate the reactivity of carboxylate leaving groups in efforts to identify carboxylate ligands with optimal pharmacological properties in a clinical environment.

To expand our series, moderately reactive hydroxyacetate and lactate are utilized and examined.

2.2 Synthesis and Characterization

The synthesis of TPA compounds is adopted and modified from a method published previously

(70) .

Figure 2.3. Synthetic scheme of TPA compounds. L= 4-picoline, isoquinoline; R=H (Formate), R=CH3

(Acetate), R=CH2OH (Hydroxyacetate), R=CHCH3OH (Lactate).

Starting Material- Cisplatin was synthesized by using a method published previously (108). All the chemicals and solvents were purchased from common vendors and used as supplied.

Preparation of trans-[Pt(L)(NH3)I2]

Cisplatin (1 mmol) was suspended in 10 mL of water and AgNO3 (2 mmol) was added to the platinum solution. A solution of planar amine (2 mmol) was prepared in methanol and then added to the platinum solution. The mixture was stirred overnight in the dark. Next day, the mixture was

23 stirred under reflux conditions for 4 h to complete the reaction. Then the hot solution was filtered through celite to remove silver chloride formed during the reaction. Excess amount of KI (3 mmol), dissolved in water, was added to the solution and the mixture was subsequently evaporated to dryness. The white crude was suspended in ethanol and heated to reflux temperature during 4 h.

Orange solid was isolated and washed with water, methanol, and ethyl ether.

1 trans-Pt[(4-pic)(NH3)I2]. H NMR((CD3)2CO): δ 8.7(d, 2H), 7.2(d, 2H), 3.7( s, 3H), 2.4(s, 3H.)

Anal. Calc’d for C6H10N2I2Pt: C 12.89, H 1.80, N 5.01. Found: C 13.08, H 1.68, N 5.04

1 trans-Pt[(isq)(NH3)I2]. H NMR((CD3)2CO): δ 9.6 (s, 1H), 8.7(d,1H), 8.3(d, 1H), 8.1(d, 1H),

7.9(d, 1H), 7.6(m, 2H). Anal. Calc’d for C9H10N2I2Pt: C 18.17, H 1.69, N 4.71. Found: C 18.32,

H 1.66, N 4.49.

Preparation of trans-[Pt(L)(NH3)(COOR)2],R=CH3, CHCH3OH.

1 mmol of trans-Pt[I2(L)(NH3)] complex was suspended in water 1.98 equivalent of freshly synthesized AgCOOR was added to the platinum solution and was stirred overnight in dark at

60 °C. The hot solution was filtered through celite to remove silver iodide formed and the solution was evaporated to dryness. White solid was recrystallized in MeOH/Ether. If the product turned brown or grey, it was dissolved it in a minimum amount of water and filtered through celite to remove any impurities.

Preparation of trans-[Pt(L)(NH3)(COOR)2], R=H, CH2OH.

2.2 mmol of formic acid was dissolved in minimum amount of water. 1 mmol of Ag2O was added to the solution. The mixture was stirred in dark for 8 h until black solid (Ag2O) was no longer present in the solution. To the solution, 1 mmol of trans-Pt[(L)(NH3)(I)2] complex was added and was stirred in dark overnight. Next day, the mixture was filtered through celite to remove AgI formed during the reaction. The filtrate was dried under vacuum and acetone added to precipitate out white solids. The product was collected through membrane filter then washed with acetone and ether to remove any free carboxylic acid. If the product turned brown or grey, they were dissolved it in minimum amount of water and filtered through celite to remove any impurities.

Figure 2.4. Structure of trans-platinum planar amine (TPA) compounds synthesized.

1 [1] H NMR(D2O): δ 8.3(d, 2H),7.45(s, 2H), 7.3(d, 2H), 2.25(s, 3H), Anal. Calc’d for C8H12N2O4Pt:

C 24.31, H 3.06, N 7.09. Found: C 24.13, H 3.06, N. 7.15

1 [2] H NMR(D2O): δ 8.3(d, 2H), 7.3(d, 2H), 2.5(s, 3H), 1.75(s, 6H). Anal. Calc’d for

C10H16N2O4Pt: C 28.37, H 3.81, N 6.62. Found: C 28.09, H 3.68, N 6.41

1 [3] H NMR(D2O): δ8.3(d, 2H), 7.3(d, 2H), 4.1(s, 4H), 2.45(s, 3H). Anal. Calc’d for

C10H16N2O6Pt: C 26.38, H 3.54, N 6.15. Found:C 26.12, H 3.23, N 6.01.

1 [4] H NMR(D2O): δ8.3(d, 2H), 7.3(d, 2H), 4.1(q, 2H), 1.1(d, 6H). Anal. Calc’d for C12H20N2O6Pt:

C 29.82, H 4.17, N 5.80. Found:C 29.72, H 4.23, N 5.91.

1 [5] H NMR(D2O): δ9.2(s, 1H), 8.3(d, 1H), 8.15(d, 1H), 7.8-8.0(m, 4H) 7.3(s,2H). Anal. Calc’d for C11H12N2O4Pt: C 30.63, H 2.80, N 6.49. Found:30.32, H 2.76, N 6.32.

1 195 [6] H NMR(D2O): δ 9.2(s, 1H), 8.3(d, 1H), 8.1(d, 1H), 7.8-8.0(m, 4H), 2.0(s, 6H). Pt NMR

(D2O): -1443. Anal. Calc’d for C13H16N2O4Pt: C 33.99, H 3.51, N 6.10. Found: C 33.95, H 3.27,

N 5.80

1 [7] H NMR(D2O): δ 9.0(s, 1H), 8.1(d, 1H), 8.0 (d, 1H), 7.9(d, 1H), 7.8(d, 1H), 7.7(t, 1H), 7.6(t,

195 1H), 3.9(s, 4H). Pt NMR (D2O): -1441. Anal. Calc’d for C13H16N2O6Pt: C 31.78, H 3.28, N

5.70. Found: C 31.61, H 2.93, N 5.64.

1 [8] H NMR(D2O): δ 9.0(s, 1H), 8.1(d, 1H), 8.0 (d, 1H), 7.9(d, 1H), 7.8(d, 1H), 7.7(t, 1H), 7.6(t,

195 1H), 4.1(q, 2H), 1.1(d, 6H). Pt NMR (D2O): -1450. Anal. Calc’d for C15H20N2O6Pt: C 34.69,

H 3.88, N 5.39. Found: C 34.22, H 3.51, N 5.26.

2.3 Evaluation of TPA Compounds by Chemical Techniques

Figure 2.5. Structures of TPA compounds studied.

To evaluate the effects of carboxylate modification on TPA compounds, chemical properties of TPA compounds including hydrolysis rate, solubility, and rate of interaction with sulfur nucleophiles are assessed in this section. Here, with an isoquinoline as a carrier ligand, moderately reactive carboxylate leaving groups, hydroxyacetate (TPA2) and lactate (TPA3), are observed in contrast to previously studied acetate compound (TPA1) to further modulate and optimize the reactivity of the compounds (Figure 2.5) (66).

2.3.1 Experimental Condition

Aqueous solubility

Solubility was measured by sequential addition of water and agitation for 15 min until solution was no longer clear. The results were determined from an average of 3 trials.

Hydrolysis rate

For hydrolysis studies, 10-3 mM complex was dissolved in 1 mL of nanopure water. The pH values of the samples were controlled in regular intervals and readjusted if necessary by addition of HNO3

(1, 0.1, 0.01 M) or NaOH (1, 0.1, 0.01 M). Aliquots of 20 mL were taken from the bulk solution for HPLC analysis. The aquated species were identified by comparison with the hydrolysis profile produced by the corresponding (labile) nitrate compounds.

Reaction with N-Acetyl-Methionine by 1H NMR Spectroscopy

Platinum compounds were incubated with N-acetyl-methionine in a reaction ratio of 1:2 (platinum

1 complex: N-acetyl-methionine) in D2O. H NMR at various time points up to 16 h was recorded on a Varian Mercury series 300 MHz NMR spectrometer using a 5 mm probe. 1H NMR spectra were referenced to sodium 3-(trimethylsilyl)-propionate (TSP) at d = 0 ppm. Approximate half- lives (t1/2) of each reaction were calculated by plotting the integral ratio of unreacted vs. reacted products. Approximate half-life is defined as time when 50% of starting material was converted to methionine bound product.

2.3.2 Results

Table 2.2. Initial hydrolysis rate, aqueous solubility, and methionine binding rate of TPA compounds. Values in parentheses are from ref (66), where the values were obtained in PBS buffer.

Aqueous Initial Hydrolysis Methionine Binding Compound -1 Solubility Rate, k1 (s ) Rate (t h) (mg/mL) 1/2, t-[Pt(formate)2(isq)(NH3)] - 1.5 (2.6) TPA1 7.4 x 10-7 6.7 20.4 (10.7) TPA2 5.7 x 10-7 1.6 10.6 TPA3 5.7 x 10-7 4.3 15.4

Aqueous solubility is one of the important parameters to achieve desired concentration of drug in a systemic circulation for achieving required pharmacological response (109,110).

Carboxylate leaving group was shown to enhance water solubility of the TPA compounds and their solubility was measured and the results are shown in Table 2.2. TPA compounds demonstrated enhanced aqueous solubility compared to the chloride analog, although it was not significantly enhanced due to presence of hydrophobic isoquinoline ligand.

The mutually weak trans influence of the carboxylate ligands resulted in significantly less reactive compounds than the chloride analogs and the reactivity of the TPA were evaluated by measuring the initial hydrolysis rate (k1) (Table 2.2). These results may be contrasted with

-4 -5 transplatin (k1 = 1.9 x 10 ) and cisplatin (k1 = 5.2 x 10 ), where the rate of hydrolysis for TPA carboxylates are significantly slower than those containing chloro ligands by 2-3 orders of magnitudes (111,112).

Figure 2.6. Structures of N-Acetyl-Methionine.

To evaluate the reactivity of TPA compounds further, substitution rates with N-acetyl– methionine (NAM) was measured. Methionine is a thioether containing amino acid that is abundant in human proteins (113,114). It is used as a probe to measure the rate of platinum drug inactivation by sulfur-containing nucleophiles.

Figure 2.7. Substitution reaction of TPA3 with N-acetyl-methionine (1:2) followed by 1H NMR spectroscopy. a. shift of the H2 of isoquinoline. b. formation of free carboxylate.

The reactions of TPA compounds with thioether containing NAM amino acid were monitored by observing the shift of the H2 proton of isoquinoline from δ = 9.03 ppm to 9.59 ppm, where the shift is a result of carboxylate leaving group substitution with NAM to form PtN2S2

(Figure 2.7a). This can be also confirmed by monitoring the shift of the protons of carboxylate leaving group from δ =1.06 (platinum bound) to δ = 1.2 (free carboxylate) (Figure 2.7b). The approximate half-lives (t1/2) of each reaction were calculated based on the peak integration of the

H2 proton of isoquinoline, and the values found were calculated to be 20.4 h, 10.6 h, and 15.4 h for TPA1, TPA2, and TPA3, respectively (Table 2.2). With previously published results (66), the rate of reactivity to NAM can be listed as the following (highest reactivity to lowest): formate > hydroxyacetate > lactate > acetate. It is interesting to note that the hydroxyacetate (TPA2) which contains a hydroxyl functional group, showed higher reactivity than the compounds with acetate

(TPA1); while the lactate (TPA3) with an additional methyl group showed a decrease in reactivity compared to the compounds with hydroxyacetate (TPA2).

2.4 Binding of TPA Compounds with DNA

DNA is the primary pharmacological target of the platinum-based anticancer agents and is considered as the most crucial factor in determining the potency of platinum chemotherapeutics, since the structural disturbance of DNA induced by platinum compounds is known to be the primary mechanism of its cell death. Transplatin was shown to induce a significant amount of

DNA platination in previous studies (115). Yet, DNA platination does not always lead to perturbation of the DNA structure since the perturbation of the DNA structure heavily depends on the type of Pt-DNA adducts formed (71,58,116).

In this section, the perturbation of DNA structure induced by

TPA compounds was evaluated by a competitive ethidium

bromide (EtBr) fluorescence assay. The fluorescent probe,

EtBr, is a DNA binding fluorescent dye that intercalates into

the minor groove of DNA with no sequence preferences

(117,118). Binding of fluorescent probe EtBr to DNA by Figure 2.8. Structure of Ethidium intercalation is blocked in a stoichiometric manner by Bromide (EtBr) structure perturbation induced by platinum compounds including cisplatin, which results in a loss

of fluorescence intensity (119,120). This competitive assay can also be used to distinguish between

perturbations induced in DNA by mono- and bi-functional adducts of platinum compounds (121),

where mono-functional adducts do not perturb the DNA structures to prevent the intercalation of

EtBr.

As a parallel experiment, the melting temperature of DNA modified by platinum

compounds measures the qualitative effect of platinum compounds on DNA thermal stability,

where cisplatin-DNA crosslinks are known to reduce the thermal stability of DNA (122,123). The

frequency and nature of Pt-DNA adducts are crucial parameters contributing to cytotoxic activity

and these biophysical assays with DNA are useful tools to predict biological consequences of the

platinum compound, thereby contributing to the rational basis for designing and screening of new

candidates for platinum chemotherapeutic agents.

Figure 2.9. Dependencies of (a). ethidium bromide fluorescence and (b). melting temperature on rb

(drug to nucleotide ratio) for DNA modified by various platinum complexes in 10 mM NaClO4 at 37 °C for 48 h. (X) cisplatin; (●) transplatin; (∆) [PtCl(dien)]Cl; (○) trans-[PtCl2(NH3)(quin)]. From Ref (124).

In a previous study, the TPA chloride analog, trans-[Pt(Cl2)(NH3)(quin)], significantly decreased EtBr fluorescence (Figure 2.9a) and melting temperature (Figure 2.9b) showing similar ability to perturb DNA structure to that of cisplatin (124). These fluorescence quenching effects were not observed with mono-functional [PtCl(dien)]Cl and transplatin due to their lack of ability to induce DNA structural perturbation (124,125). To assess DNA structural perturbation induced by TPA compounds to cause DNA structural perturbation, TPA compounds synthesized in this chapter were evaluated by using EtBr competitive and DNA melting temperature assay.

2.4.1 Experimental Conditions

Ethidium Bromide Fluorescence Quenching Assay

Calf Thymus DNA (100 μM) was incubated with the compounds for 48 h at 37 ºC followed by treatment with ethidium bromide (EtBr) at ri values from 0.05 to 0.25. The experiments were 33 carried out in 10 mM NaClO4 in the presence of 0.4 M NaCl to avoid secondary binding of the

EtBr to DNA (121,124). Samples were irradiated with 525 nm light and emission was recorded at

600 nm with a scan rate of 600 nm/min at 25 ºC. The results were determined from an average of

3 trials.

Melting Temperature of CT-DNA Modified with TPA compounds

200 μM calf thymus DNA samples (expressed as nucleotides) were incubated with the platinum complexes at various ratios (ri= 0 to 0.05; ri being the ratio of drug per nucleotide) in 10 mM

NaClO4 at 37 °C for 24 h. To avoid the formation of air bubbles during the experiment the samples were degassed by stirring under vacuum for 5 min. The absorbance at 260 nm was recorded while the temperature was increased from 40 or 60 to 95 °C at a rate of 0.5 °C/min. A low headspace cuvette with 10 mm path length filled with 150 μL sample was used to minimize solvent evaporation. The resulting melting curves were normalized and the melting temperature was determined by sigmoidal curve fitting using Microsoft Excel. Experiments were performed in duplicate.

2.4.2 Results

Figure 2.10. Quenching of ethidium bromide fluorescence intensity upon treatment with platinum compounds after 48 h incubation.

Figure 2.11. DNA melting temperature of TPAs. 200 μM calf thymus DNA samples (expressed as nucleotides) were incubated with the platinum complexes at various in 10 mM NaClO4 at 37 °C for 24 h.

EtBr assay with TPA compounds showed a minor loss in fluorescence intensity while cisplatin showed significantly higher loss of the fluorescence intensity (Figure 2.10). These results were consistent with DNA melting temperature assay (Figure 2.11), where the melting temperature of DNA did not significantly alter after the treatment with TPA compounds. From these results, it is suggested that the rates of DNA perturbation structure caused by TPA compounds were much slower than cisplatin and trans-[Pt(Cl2)(NH3)(quin)] due to enhanced stability of the drug. This similar effect was previously seen with carboplatin (126).

A combination of chemical experiments showed that TPA lactate and hydroxyacetate compounds exhibited moderate reactivity in comparison with previously utilized acetate and formate. Biophysical studies of the TPA compounds synthesized in this chapter with DNA showed that they do not readily perturb the structure of DNA, which may be a result of their enhanced stability. Cellular consequences of the modulated reactivity are investigated in the next chapter by assessing their biological effects in cells.

2.5 Contributions

All of the work in this chapter was done by Daniel E. Lee with exception of hydrolysis rate and solubility rate, which were done by Erin Ma.

Chapter 3

Biological Effect of TPA

Compounds

CHAPTER 3. BIOLOGICAL EFFECT OF TPA COMPOUNDS

In the previous chapter, the reactivity of TPA compounds were modulated by modifying the structures of the carboxylate leaving group. To develop a clinically relevant platinum-based chemotherapeutic which can circumvent cisplatin resistance, it is crucial for the compound to exhibit cytotoxic activities and high cellular internalization while demonstrating enhanced stability to metabolic inactivation by sulfur nucleophiles. To examine these biological parameters and to understand the biological consequences of modulated TPA reactivity, biological assays including cytotoxicity, cellular accumulation, and cell cycle effects were evaluated.

Figure 3.1. MTT assay to measure cell survival and proliferation.

Cytotoxicity is considered as a primary criterion to measure potency of platinum chemotherapeutics. The effects of TPA on cell proliferation were measured by using MTT assay in tumor cells. This yellow tetrazole is reduced to violet formazan through mitochondrial reductase in living cells and is commonly used to as a quantitative colorimetric assay for mammalian cell survival and proliferation (127,128). The potency is commonly expressed as IC50, or the drug concentration required to inhibit 50% of the maximal cell growth.

Cellular accumulation, which is a net measure of influx and efflux, is another critical criterion contributing to cytotoxic activity (129,130). It is also worthwhile to mention that acquired and inherent resistance to traditional platinum compounds involves reduced cellular accumulation, which could result from increased efflux, reduced influx, and/or degradation into inactive metabolites by sulfur nucleophiles (68,131,132). The cellular accumulation of platinum based drug can be measured by treating cells with the platinum compounds and measure the amount of platinum in cells by using atomic absorption or inductively coupled plasma- mass spectroscopy for quantitative analysis.

Another biological effect to investigate is cell cycle effects induced by TPA compounds. It is well recognized that DNA damage induced by cisplatin usually activates cell cycle checkpoints, which inhibit cell cycle progressing, where the inhibition results in depletion of G1 phase and S phase accumulation in cisplatin-sensitive ovarian carcinoma A2780 cells (133,134,135,136).

Previously, TPA compounds retained their activities in cisplatin-resistant and p53 mutant cells, which may indicate that their toxicity might be mediated by mechanisms differ from cisplatin. To confirm this, the cell cycle effects induced by TPA compounds were examined in this chapter.

3.1 Cytotoxicity, Cellular Accumulation, and Metabolic Stability of TPA

Compounds

3.1.1 Experimental

Cell Culture

The human ovarian cancer cell line, A2780 was grown in monolayer culture in RPMI 1640

(Invitrogen, Grand Island, NY, USA; Cat# 11875-093) supplemented with 10% fetal bovine serum

(FBS, Quality Biologicals, Gaithersburg, MD, USA; Cat# 110-001-101US) and 100 U/mL penicillin and 100 mg/mL streptomycin (Invitrogen, Grand Island, NY, USA; Cat# 15140), at

37 °C in a 5% CO2 incubator.

Growth Inhibition Assay

Five thousand cells/well in 100 µL were seeded in a 96-well plate and allowed 24 h to attach.

A2780 cells were then treated with the TPA compounds for 72 h. Following TPA compound removal, the cells were incubated in the presence of 0.5 mg/mL MTT reagent [(3,4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Sigma–Aldrich, St. Louis, MO, USA;

Cat# M2128] in RPMI medium for 3 h at 37°C. The MTT reagent was removed and 100 µL DMSO was added to each well. The plate was then incubated on a shaker at room temperature in the dark.

The spectrophotometric reading was taken at 570 nm using a microplate reader. For the time dependent serum incubation studies, 1 mM of each Pt compound was incubated in FBS for 0, 24 and 48 h (final concentration of 500 μM). The cells were then treated with the serum pre-incubated compounds at a concentration range of 0.78 – 50 μM for 24 h.

Cellular Accumulation of Platinum Compounds

A2780 cells were treated with 10 µM of platinum compounds following incubation with or without serum for 48 h. After 12 h treatment with each compound, cells were harvested. To digest samples,

1 mL of concentrated HNO3 was added to the pellet and left to digest for three days. 2 mL of water was then added to dilute the samples and the solutions were filtered through a 0.45 μGHP filter, and analyzed on ICP-MS to determine the platinum concentration. Error bars indicate Mean ±

S.E.M. (the standard error of the mean) from two independent experiments.

3.1.2 Results

Figure 3.2. Cytotoxicity curve of TPA compounds and cisplatin before and after incubation with serum in A2780 cells. Platinum compounds were incubated with fetal bovine serum for 24 h and 48 h then treated cells for 72 h. Growth inhibition was then measured by MTT after drug treatment.

Table 3.1. Cytotoxicity of TPA compounds and cisplatin before and after incubation with serum in A2780 cells.

IC50 (μM) 24 h Serum 48 h Serum Fold Increase in Compound Control Incubation Incubation IC50 (48 h) Cisplatin 1.08 ± 0.1 7.95 ± 0.07 28.3 28 TPA1 23.5 ± 1.5 31.1 ± 3.7 36.1 1.5 TPA2 11.4 ± 0.9 13.5 ± 2.8 16.4 1.6 TPA3 14.8 ± 3.1 16 ± 0.5 21.2 1.4

Figure 3.3. Structures of TPA compounds studied.

Therapeutic efficacy of platinum chemotherapeutic agents is reflected as a balance between metabolic inactivating interactions and biological consequences of DNA toxicity; therefore, cytotoxic activity and metabolic stability were measured to assess the efficacy of TPA compounds as potential new chemotherapeutic agents (137). To assess the metabolic stability of TPA compounds with modified carboxylates, IC50 values for the TPA compounds were obtained by using MTT assay in A2780 ovarian carcinoma since ovarian cancer cells are known to be sensitive to platinum chemotherapeutics treatments. All of the TPA compounds exhibited low micromolar cytotoxicity similar to that of cisplatin as shown in Figure 3.2. Among all, the highest cytotoxicity was exhibited by most reactive TPA2. TPA1, which was shown to be more inert in the substitution reaction with n-acetyl methionine (NAM), exhibited less cytotoxicity activities by requiring approximately two fold higher drug concentration than TPA2 and TPA3.

To evaluate the metabolic stability of TPA compounds, the cytotoxicity assays were also performed after 24 h and 48 h incubation with fetal bovine serum (FBS). Serum is a liquid portion

42 of blood and sulfur containing molecules responsible for the platinum drug inactivation are abundant in serum (29). While cisplatin showed diminished cytotoxicity upon serum incubation with an increase in IC50 value by 28 fold from 1.1 µM to 28.3 µM, TPA compounds retained their cytotoxic activities significantly more effectively with less than 2 fold increase in IC50 (Figure 3.2).

Although the kinetic studies with NAM showed that TPA2 and TPA3 were more reactive than

TPA1, their stability in serum were not significantly different among these compounds. From these results, TPA2 and TPA3 demonstrated superior cellular efficacy by demonstrating higher cytotoxicity activities while demonstrating similar metabolic profiles.

Cellular Accumulation

Figure 3.4. Cellular accumulation of platinum compounds in A2780 cells. Cells were treated for 12 h with 10 µM of platinum compounds following incubation with or without serum for 48 h. After the treatment with each compound, cells were harvested and platinum uptake was measured on Varian ICP-MS. Error bars indicate Mean ± S.E.M. from two independent experiments.

Intracellular accumulation of platinum compound is one of the key factors contributing to their cytotoxic activities. Decreased intracellular concentration is a known cause of cisplatin resistance; thus, the intracellular concentration resulted from the treatment with TPA compounds were assessed by using ICP-MS. Treatments of A2780 cells with 10 μM of cisplatin resulted in intracellular platinum level of 45 attomole Pt Cell-1, while compounds generally demonstrated higher cellular accumulation by resulting in 47 (TPA1), 119 (TPA2), and 89 (TPA3) attomole Pt cell-1. TPA3 was speculated to show the highest accumulation due to presence of additional methyl group on lactate, thereby contributing added hydrophobicity to the compound; however, the most reactive TPA2 demonstrated highest intracellular platinum level among all compounds studied

(Figure 3.4)

Cellular accumulation was also measured after 48 h serum treatment since metabolic inactivation is a known cause of the reduced accumulation (138,139). Intracellular platinum levels of cisplatin decreased by 4.6 fold after serum incubation while TPA compounds demonstrated no significant loss in intracellular platinum levels after the incubation (Figure 3.4). This is in agreement with the metabolic stability of TPA compounds that were seen in cytotoxicity studies.

Interestingly, TPA2, the most reactive compound in this study, demonstrated highest intracellular platinum concentration before and after the serum incubation. TPA2 demonstrated highest cytotoxic activity and cellular accumulation while demonstrating similar stability profile to metabolic inactivation in comparisons with other TPA compounds; therefore, a combination of biophysical and biological assays indicate that TPA2 has the most promising pharmacological properties amongst the compounds examined.

3.2 Cell Cycle Effects of TPA Compounds

3.2.1 Experimental

Analysis of Cell Cycle Distribution

A2780 cells were seeded in 10-cm tissue culture dishes at a density of 5 X 105 cells per dish. After

24 h, cells were treated with an equimolar dose of platinum compounds (10 µM) for 24 and 48 h.

Both attached and ﬂoating cells were harvested at the different time points. Cells were stained with

DNA intercalating fluorescent probe, propidium iodide, and analyzed using flow cytometry as described previously (140,141).

3.2.2 Cell Cycle Effects

Figure 3.5. Effects of treatment with platinum compounds on cell cycle. A2780 cells were treated with 10 µM of each platinum compounds for 48 h. Cells were then harvested, stained with propidium iodide, and then analyzed using flow cytometry.

In a previous study reported by Fojo et al, cell cycle effects were examined with a series of

TPA compounds in KB-3.1, KBCP20 (cisplatin-resistant), and KBOX60 (oxaliplatin-resistant) epidermoid carcinoma cells. The results showed that TPA compounds did not induce any significant cell cycle effects (80). To confirm this result, cell cycle effects were evaluated in A2780 cells with TPA compounds synthesized in this chapter. After 48 h treatment, cisplatin disrupted the cell cycle by slowing cell progression through S-phase in response to DNA damage (Figure

3.5). In contrast, a cytotoxic concentration of the TPA compounds did not significantly alter the cell cycle, although a rise in sub-G1 indicating apoptotic activity was present upon treatment with

TPA compounds. The minimal influence in cell cycle may suggest that TPA compounds caused cell death by a different mechanism than cisplatin, which may explain why TPA compounds showed unaffected activity in cisplatin-resistant cells.

3.3 Possible Cellular Targets Other Than DNA

Figure 3.6. Bifunctional DNA crosslinks and DNA-protein cross-links induced by TPA. From ref (77).

3.3.1 Ternary Topoisomerase I-DNA Interaction

Our results showing the minimal influence in cell cycle and the lack of perturbation in

DNA structure raise a question whether DNA is the only (or major target) of the TPA compounds responsible for the cell death induced by the TPA compounds. While relatively few studies were performed on the cellular effects of TPA, previous biochemical studies suggest that its cellular pharmacology involves four distinct types of DNA lessions: monofunctional DNA adducts, bifunctional DNA adducts, ternary DNA-DNA crosslinks, and ternary DNA-protein crosslinks

(9,78,97,142). Bifunctional intra-strand and inter-strand DNA adducts induced by platinum compounds are known to cause significant DNA structural perturbation; however, trans isomers are sterically restricted to form these adducts predominantly.

One interesting study revealed that treatment of MCF-7 (breast cancer cells) with trans-

[PtCl2(NH3)(tz)] and trans-[PtCl2(py)2]] resulted in a formation of DNA-topoisomerase I adduct similar to that of camptothecin, a known topoisomerase I inhibitor (77). DNA topoisomerases are essential enzymes that relieve the torsional strain by concerted breakage and rejoining of DNA strands and have been recognized as a target for anticancer treatment (143). In contrast, this effect was not evident with the cis isomer at the same concentrations. It is noteworthy to mention that the formation of topoisomerase I-DNA upon treatment with trans-[PtCl2(NH3)(tz)] coincided with the promotion of apoptosis while cisplatin treatment did not produce either topoisomerase I-DNA complexes nor apoptosis (77). Although further details have to be studied, these findings suggest that the unique platinum-DNA adduct structure induced by TPA compounds can lead to significantly different cellular downstream effects such as protein recognition and apoptotic pathways, which may provide an explanation of their unaffected activities in cisplatin-resistant cells.

3.3.2 Zinc-Finger Proteins

A zinc-finger is a small protein structural motif that is characterized by the coordination of zinc ions with a combination of cysteine and histidine to stabilize the fold (144). Their functions are diverse and they have become important cellular targets for anti-cancer and HIV treatments

(145,146). Platinum (II) compounds have a strong tendency to bind to sulfur or nitrogen containing proteins, and zinc-finger motifs with cysteine and histidine have been investigated as possible targets for the platinum compounds. The interaction of zinc-finger with trans-platinum compounds was initially exemplified by trans-platinum mononucleobase compounds, where these compounds interacted with the C-terminal finger of HIV NCp7 protein and resulted in zinc ejection from the peptide, accompanied by loss of tertiary structure and its function (147).

A further zinc-finger target for TPA compounds was mentioned as zinc-finger Sp1 (148).

The human transcription factor Sp1 is a C2H2 zinc-finger protein that regulate gene expression by binding to specific DNA sequences and they are known to have a pivotal role in various cellular processes including tumorigenesis (149). trans-[PtCl2(NH3)(tz)] exhibited significant reactivity to Sp1 while cisplatin did not show any detectable effect on Sp1. It is noteworthy to mention that transplatin has shown much less affinity to Sp1, which suggests that thiazole ligand is a crucial determinant for the high affinity that was shown with trans-[PtCl2(NH3)(tz)]. The platination of

Sp1 disrupts the secondary structure of the protein and interferes with the DNA-binding function of Sp1 (148). This interference with zinc-finger motifs may down-regulate the expression of some oncogenes and these results revealed a novel approach for the inhibition of Sp1, suggesting another possible target responsible for the antitumor activity of TPA compounds.

3.3.3 Telomerase

Telomerase was revealed as another possible target for TPA compounds in a study (150).

Telomeres are protective caps at the ends of human chromosomes, which shorten with each cell division in normal cells whereas, in tumors, they are continuously lengthened by telomerase enzymes. Telomerases are overexpressed in most of the cancers while they are present in very low concentration in normal cells and it is recognized as a potential target for antitumor agents to inhibit tumor cell proliferation (151). Upon treatment of MCF-7 cells with trans-[PtCl2(py)2], telomerase activity was significantly inhibited in the first 24 h, while its cis isomer, cis-[PtCl2(py)2], did not show any inhibition. The observation that trans-[PtCl2(py)2] induced a rapid decrease of telomerase activity within 24 h suggests that trans-[PtCl2(py)2] interacts with existing telomerase rather than preventing the telomerase synthesis (150). Although telomere length reduction seemed not to be the main mechanism of the observed cell apoptosis induced by trans-[PtCl2(py)2], a rapid and direct interaction with the existing pool of telomerase reveals another unique cellular feature of TPA compounds.

3.3.4 Nuclear Protein Positive Cofactor PC4

An MS-based proteomic strategy utilizing DNA-conjugated gold nanoparticle probes recently revealed that the human nuclear protein positive cofactor 4 (PC4) selectively binds to cross-linked DNA by trans-[PtCl2(NH3)(tz)], implying that PC4 may play a role in cellular response to DNA damage by trans-[PtCl2(NH3)(tz)] (152). This strategy was developed and validated by a successful identification and quantification of HMGB1 (high mobility group protein

B1) upon cisplatin treatment, which is well known protein that interacts selectively with 1,2-cross- linked cisplatin-DNA adduct (152,153). The validated MS-based proteomic strategy was applied

49 and identified the cellular proteins in response to DNA damage induced by trans-[PtCl2(NH3)(tz)].

Among the proteins identified, PC4 was most selective to the damaged DNA caused by trans-

[PtCl2(NH3)(tz)] (152). The multifunctional nuclear protein positive cofactor 4 (PC4) is involved in various cellular processes including transcription, replication and chromatin organization; and was also found to have a crucial role in the early response to DNA damage (154). Thus, the specific interaction of DNA damaged by trans-[PtCl2(NH3)(tz)] and PC4 may be related to the unique anticancer activity of trans-[PtCl2(NH3)(tz)] and further studies are deserved to understand the unique cell death mechanism induced by TPA compounds.

3.4 Conclusion

The development of chemotherapeutic agents that can circumvent cisplatin resistance has been a major ongoing strategy in platinum research. Previously, we have seen a promising cytotoxic activity in cisplatin-resistant cells with TPA compounds. The major mechanism of cisplatin resistance is known to be metabolic inactivation by sulfur molecules, reduced accumulation, and enhanced Pt-DNA adduct recognition and repair. Thus, a combination of systematic chemical (Section 2.3), biophysical, and biological properties were assessed to address this issue with further optimized TPA compounds. From these results, we propose TPA2 as a lead candidate for further development and examination to circumvent the existing cisplatin resistance, where it demonstrated low micromolar cytotoxicity, enhanced metabolic stability, higher cellular accumulation, and distinct cell cycle effects than cisplatin. From the DNA binding profile and cell cycle effects induced by TPA compounds, it is tempting to speculate that other pharmacological targets besides DNA may be responsible for the cell death caused by TPA; thereby, contribute to their retained activity in cisplatin resistant cells. Further detailed mechanistic studies are deserved

50 for a deeper understanding of the unique cytotoxic activity of TPA, which will provide a valuable insight to the development of a clinically relevant TPA compound.

3.5 Contributions

Cytotoxicity and cell cycle effects were done by Vijay Menon.

Daniel E. Lee and Vijay Menon equally contributed to cellular accumulation study.

All of other work in this chapter was done by Daniel E. Lee.

Chapter 4

Synthesis and Characterization

of Polynuclear Platinum

Compounds

CHAPTER 4. SYNTHESIS AND CHARACTERIZATION OF

POLYNUCLEAR PLATINUM COMPOUNDS

Figure 4.1. BBR3464 and its noncovalently binding analogues, AH44 and Triplatin NC

Despite its success demonstrated in pre-clinical and Phase I clinical studies, Phase II clinical studies discouraged the usage of BBR3464 due to a rapid degradation of BBR3464 into inactive metabolites (93,94). Its noncovalently binding analogues, AH44 and Triplatin NC, have overcome the pharmacokinetic problems by binding with DNA solely through noncovalent electrostatic interactions, which was accomplished by replacing chloride leaving group with inert ammine (NH3) or “dangling amine” (NH2(CH2)6NH3). These highly charged platinum compounds introduced a novel mode of DNA binding by interacting with negatively charged phosphate backbone of DNA to cause DNA perturbation. With the distinct feature, they exhibited superior biological profile including low micromolar cytotoxicity in tumor cells, high cellular accumulation and unaffected cytotoxicity in cisplatin resistant and p53 mutant cells (Figure 4.2 and Table 4.1). Interestingly,

Triplatin NC (8+) exhibited higher cytotoxicity and cellular accumulation than AH44 (6+) in various tumor cells by up to 20 fold (103). It is unclear why the higher charged compound with dangling amine ligand exhibited these superior pharmacological properties and further

53 investigation on structural-activity relationship are established in this chapter which is accomplished by the synthesis of a novel series of noncovalently binding polynuclear platinum compounds (PPC-NC) with varied number of platinum centers and the overall charge of the compound.

Table 4.1. Cytotoxicity of cisplatin (c-DDP), BBR3464, AH44 (I), and Triplatin NC (II) in various tumor cells. From ref (103).

Figure 4.2. Cellular accumulation of BBR3464, AH44 (I), and Triplatin NC (II). From ref (103).

In efforts to establish a structure-activity relationship of PPC-NC, a series of dangling amine

PPC-NC with different (mono-, di-, tri-, tetra-) number of platinum centers, resulting in differences in the overall charge and the length of the platinum compounds. It is important to note that AH44

(trinuclear, 6+) was studied with this series to directly compare between Diplatin NC (dinuclear,

6+) and Triplatin NC (trinuclear, 8+) to understand the biological consequences of the utilizing different inert ligand (dangling amine vs ammine) to the PPC-NC.

Figure 4.3. Inhibition of heparinase I by cisplatin, PPC, and R9. From Ref (155).

Positive charge of PPC-NC is thought to be responsible for its higher cellular internalization, where the mechanism involves binding to negatively charged heparan sulfate proteoglycans

(HSPG) (156). Recently, the strong binding of PPC-NC to HSPG showed inhibition of enzymatic cleavage of the glycans by heparinase (155). Cisplatin did not inhibit cleavage, consistent with the fact that it is not a substrate for HSPG-mediated internalization (Figure 4.3) (156). Heparinase is an enzyme that is known to cleave HSPG and it is over-expressed in tumors and there is significant

55 correlation between metastatic potential and heparinase activity (157,158). When HSPG are degraded by the enzyme, it releases angiogenic and growth factors leading to cell migration, growth, and angiogenesis (159,160,161); thus, the strong binding of PPC to HSPG to protect against enzymatic cleavage opens a new approach to glycan targeting for antiangiogenic effects.

Yet, structure-activity relationship remains undefined. A series of PPC-NC synthesized in this chapter can be used to further explore the antiangiogenic effects exhibited by PPC-NC in parallel to their cytotoxic abilities.

Figure 4.4. Proposed target structures of PPC-NC (with dangling amine) and AH44 (with NH3).

4.1 Synthetic Considerations

4.1.1 Synthetic Considerations of PPC Chloride Analogs (PPC-Cl)

Figure 4.5. 1H NMR spectra of different BBR3464 synthetic methods. A. 1H NMR spectrum of impure BBR3464 resulted from previously published method (162,163). B. 1H NMR spectrum of pure BBR3464 obtained with new synthetic approach.

A critical step in the synthesis of the BBR3464 is the coupling of the central platinum unit with the terminal platinum moieties (transplatin). In order to enable this conjugation, the amine linkers of the central platinum unit have to be deprotonated. A previously utilized method (162,163) to synthesize BBR3464 may result in an impurity involving protonated linker on the central platinum (shown in Figure 4.5A, peak d). This prevents it from coupling the terminal platinum unit to achieve the desired product and this impurity is extremely difficult to remove due to similarities in solubility with the final product. To prevent this occurrence, an alternative synthetic approach was developed to obtain the product more efficiently. The previous methods utilized an equimolar of sodium hydroxide and transplatin to achieve the final product. One major challenge in this synthetic approach was an occurrence of platinum reduction (Pt2+ → Pt0) in the presence of the strong base, sodium hydroxide, which imbalances the stoichiometry of the reaction, resulting in impurities. To prevent the platinum compounds from reducing, the non-nucleophilic base,

DIPEA (N,N- diisopropylethylamine), was utilized. In addition, rather than utilizing exact stoichiometric conditions, the new method utilizes slight excess of base and transplatin to ensure the reaction to complete, where excess materials were removed by recrystallizing in water/methanol once the reaction is complete.

4.1.2 Synthetic Considerations of PPC-NC

Figure 4.6. A comparison of 1) previous approach from ref (103) and 2) new synthetic approach to synthesize Triplatin NC.

Figure 4.7. New synthetic scheme of PPC-NC. a. 2 eq. AgNO3 then 2.2 eq. n-Boc-1,6-hexanediamine in DMF, b. 0.1M HNO3. c. 1 eq. AgNO3 then 0.5 eq hexanediamine in DMF. d. 2.2 eq. DIPEA e. mono- activated transplatin, t-[Pt(NH3)2(Cl)(NO3)].

Previously published methods to synthesize Triplatin NC involve the coupling of intermediate i, t-[PtCl(NH3)2(NH2(CH)6NHBOC)]NO3, to the central platinum moiety (Figure 4.6, approach 1). The intermediate i can remain as an impurity if the exact stoichiometry is not achieved; moreover, the solubility of i and the final product are very similar and they are extremely difficult to separate. Due to those reasons, the synthesis of PPC-NC remained extremely challenging. To overcome this difficulty, PPC-Cl were synthesized as intermediates, which was accomplished by reacting mono-activated transplatin i to the deprotonated central platinum moiety. Then boc- protected dangling amine (N-Boc-1,6-hexanediamine) was attached to the PPC-Cl, followed by

59 boc-deprotection to obtain the final product, where the residual ligand was removed by washing the final product with methanol and acetone (Figure 4.6, approach 2). This novel synthetic strategy can be applied to synthesize other compounds in the PPC-NC series as shown in Figure 4.7 and this strategy successfully allowed the desired product to be obtained without further separation/recrystallization. The obtained compounds were characterized by 1H NMR spectroscopy (for the detection of residual ligands), 195Pt NMR spectroscopy (for the detection of

PPC-Cl), and elemental analysis (CHN) to ensure that the pure products were obtained.

4.2 Synthesis and Characterization of PPC-Cl

Figure 4.8. Structure of synthesized PPC-Cl.

Starting Materials – All chemicals and solvents were purchased from common vendors. Dry

DMF was prepared by storing over 4 Å molecular sieves. Monoactivated transplatin was produced by stirring transplatin with AgNO3 overnight in DMF at room temperature in the dark. AgCl was removed by filtration.

Transplatin- Transplatin was prepared by using a method published previously (164).

1,1/t,t- 1,1/t,t was prepared using a methods published previously (46,165).

1 Yield- 83% H NMR (D2O): δ 2.7ppm (2H, t), 1.7ppm (2H, m), 1.4 (2H, m). Elemental Anal

195 calc’d for C6H28Cl2N8O6Pt2- C 9.37, H 3.67, N 14.56. Obtained- 9.73, 3.36, N 14.19. Pt NMR

(D2O): -2418 ppm

4.2.1 Synthesis of BBR3464 by New Approach

1 mmol of Monoplatin NC dissolved in DMF, 2.2 mmol of DIPEA was added. 2.2 mmol of mono- activated transplatin was added to the solution and the mixture was allow react for 2 days at ambient temperature. The solution was evaporated to dryness under reduced pressure and was dissolved in minimum amount of water. The reaction was filtered through syringe filter to remove any unreacted starting material. The filtrate was evaporated to ~1mL under reduced pressure and

25mL of acetone was added to precipitate the product. The precipitant was collected through filter and was washed with acetone.

1 BBR3464- (Yield: 42%). H NMR (D2O): δ 2.7ppm (8H, m), 1.7(8H, m), 1.4 (8H, m). Elemental

Anal calc’d for C12H50Cl2N14O12Pt3- C 11.64, H 4.07, N 15.83. Obtained- C 11.66, H 3.52, N

195 15.31. Pt NMR (D2O): -2425, -2679 ppm.

4.2.2 Synthesis of Tetraplatin-Cl

To 1 mmol of Diplatin NC (prepared as reported in section 4.3) dissolved in DMF, 2.2 mmol of

DIPEA was added. 2.2 mmol of mono-activated transplatin was added to the solution and the mixture was allowed to react for 2 days at ambient temperature. The solution was evaporated to dryness under reduced pressure and was dissolved in minimum amount of water. The reaction was filtered through syringe filter to remove any unreacted starting material. The filtrate was evaporated to ~1mL under reduced pressure and 25mL of acetone was added to precipitate the product. The precipitant was collected through filter and was washed with acetone.

1 Tetraplatin- (Yield: 46%). H NMR (D2O): δ 2.7ppm (12H, m), 1.7(12H, m), 1.4 (12H, m).

Elemental Anal calc’d for C18H72Cl2N20O18Pt4- C 12.66, H 4.25, N 16.40. Obtained- C 13.04, H

195 4.20, N 16.36. Pt NMR (D2O): -2405, -2663 ppm.

4.3 Synthesis and Characterization of PPC-NC

4.3.1 Synthesis of Monoplatin NC

Monoplatin-Boc, trans-[Pt(NH3)2(NH2(CH2)6NHBOC)2](NO3)2

A mixture of trans-diamminedichloroplatinum (1 mmol) and AgNO3 (1.98 mmol) in dry DMF was stirred overnight protected from light. The following morning, formed AgCl was filtered off.

A solution of the mono-BOC-protected hexanediamine (2.2 mmol) in dry DMF was added to the filtrate. The mixture was stirred for 3 days protected from light at ambient temperature. The solvent was then removed under reduced pressure. The residue was then dissolved in 100 ml of water at

50 °C and filtered through a membrane filter to remove any reduced silver and platinum. The filtrate was evaporated to dryness and the obtained white solid was suspended and washed with

1 acetone/methanol to remove any unreacted starting material. Yield- 74.3%. H NMR(D2O): δ

3.05(t, 4H), 2.65(t, 4H), 1.60 (m, 8H), 1.4 (m, 26H).

Monoplatin NC, trans-[Pt(NH3)2(NH2(CH2)6NH3)2](NO3)4

1 mmol of the Monoplatin-Boc was suspended in a mixture of 25 ml 0.5 M HNO3 and stirred for

2 days at room temperature. The mixture was filtered through celite to remove any unreacted starting materials and reduced silver/platinum. The volume of the filtrate was reduced to almost dryness and acetone was added to force the precipitation of the product. The formed precipitate

1 was filtered off then washed with acetone. Yield- 92.3%. H NMR(D2O): δ 3.01(t, 4H), 2.65(t,

195 4H), 1.60 (m, 8H), 1.4 (m, 8H). Pt NMR (D2O): -2653 ppm. Anal. Calc’d for C12H40N10O12Pt:

C 20.25, H 5.67, N 19.68. Found: C 20.21, H 5.60, N 19.62

4.3.2 Synthesis of Diplatin NC, Triplatin NC and Tetraplatin NC

Diplatin NC-Boc, Triplatin NC-Boc, and Tetraplatin NC-Boc

To 1 mmol of PPC-Cl (Diplatin, Triplatin, Tetraplatin) dissolved in DMF, 1.98 equivalents of silver nitrate was added. The reaction was allowed to stir for 16 h protected from light. Next day,

AgCl formed during the reaction was filtered through celite. To the filtrate, 2.2 equivalents of n-

63 boc-1,6-hexanediamine was added. The reaction was allowed to stir for 2 days at room temperature.

The solution was evaporated to dryness under reduced pressure. Then 100 ml of water was then heated to 60 ºC to dissolve the product. Dissolved product was filtered through celite to remove any reduced platinum and silver. The filtrate was evaporated to dryness and acetone was added to precipitate the product. The precipitant was collected through filter and washed with acetone.

1 Diplatin NC-BOC- Yield: 64%. H NMR(D2O): δ 3.05(t, 4H), 2.65(t, 8H), 1.60 (m, 12H), 1.4

(m, 30H).

1 Triplatin NC-BOC- Yield: 58%. H NMR(D2O): δ 3.02(t, 4H), 2.63(t, 12H), 1.62 (m, 16H), 1.4

(m, 34H).

1 Tetraplatin NC-BOC- Yield: 52%. H NMR(D2O): δ 3.05(t, 4H), 2.64(t, 16H), 1.60 (m, 20H),

1.4 (m, 38H).

Diplatin NC, Triplatin NC, and Tetraplatin NC

0.1 mmol of complex Di-, Tri, and Tetraplatin NC-Boc were suspended in 25 ml aqueous 0.1 M

HNO3 and stirred for 4 days at 50 °C. At the end of the reaction the suspension had turned into a clear almost colorless solution. 1H NMR was utilized to confirm the completion of the boc- deprotection. The solution was filtered through celite to remove any unreacted starting material.

The filtrate was evaporated to almost dryness and acetone was added to force the precipitation of the product. The residue was collected through filter and washed with methanol and acetone. If further purification was necessary, the crude product was recrystallized in water.

Figure 4.9. 1H NMR spectra of A) Monoplatin NC, B) Diplatin NC, C) Triplatin NC, D) Tetraplatin NC. A, C, D were obtained by 300MHz NMR while B was obtained by 600MHz NMR.

1 Diplatin NC- Yield- 93%. H NMR (D2O): δ 3.0 (t, 4H), 2.65 (m, 8H), 1.65 (m, 12H), 1.37 (m,

12H). Elemental Anal calc’d for C18H62N16O18Pt2- C 18.31, H 5.29, N 18.98. Obtained- C 18.51,

195 H 5.27, N 18.47. Pt NMR (D2O): -2651 ppm

1 Triplatin NC- Yield- 84%. H NMR (D2O): δ 3.0 (t, 4H), 2.65 (m, 12H), 1.65 (m, 16H), 1.37 (m,

16H). Elemental Anal calc’d for C24H84N22O24Pt3- C 17.47, H 5.13, N 18.67. Obtained- C 17.27,

195 H 5.02, N 18.21. Pt NMR (D2O): -2652 ppm

1 Tetraplatin NC- Yield- 84%. H NMR (D2O): δ 3.0 (t, 4H), 2.65 (m, 16H), 1.65 (m, 20H), 1.37

(m, 20H). Elemental Anal calc’d for C30H106N28O30Pt4- C 17.00, H 5.04, N 18.50. Obtained- C

195 16.58, H 4.62, N 17.98. Pt NMR (D2O): -2652 ppm

4.4 Contributions

All of the work in this chapter was solely done by Daniel E. Lee.

Chapter 5

DNA Binding Profiles and

Cellular Effects of Polynuclear

Platinum Compounds

CHAPTER 5. DNA BINDING PROFILES AND CELLULAR EFFECTS

OF POLYNUCLEAR PLATINUM COMPOUNDS

Figure 5.1. Structure of PPC-NC studied in this chapter.

Noncovalently binding polynuclear platinum compounds (PPC-NC) introduced a new mode of DNA binding by interacting with negatively charged phosphate backbone of DNA. Furthermore,

PPC-NC circumvented the pharmacokinetics of covalently binding platinum compounds by demonstrating inertness to metabolic inactivation by sulfur nucleophiles, while demonstrating low micromolar cytotoxicity and high cellular accumulation. To further investigate and establish the

68 structure-activity relationship (SAR) of PPC-NC, various PPC-NC with different charges and numbers of platinum centers were synthesized and characterized, as described in the previous chapter. In this chapter, a combination of biophysical and biological assays were utilized to investigate the effects of structural variation on DNA binding, cytotoxicity, and cellular accumulation. The suggested cellular entry mechanism of PPC-NC are also reported in this chapter.

5.1 Determination of Platinum Compound-DNA Interactions

5.1.1 Background

Figure 5.2. Hydrogen Bonds between Triplatin NC and DNA. From Ref (98). (A) View perpendicular to the helical axis. (B) View along the helical axis. Atoms are colored by type with carbon (DNA), green; carbon (Triplatin NC) gray; nitrogen, blue; oxygen, red; platinum, yellow.

Phosphate Clamp

Replacement of the labile chloride leaving groups with kinetically inert NH3 ligands introduced an entirely novel class of noncovalent polynuclear platinum compounds (PPC-NC),

69 where interactions with DNA are through electrostatic and hydrogen-bonding noncovalent interactions exclusively (101,166). Crystallographic characterization of DNA with these inert cationic compounds revealed that PPC-NCs do not intercalate nor do they bind in the major or minor groove; however, they interact with negatively-charged phosphate oxygen atoms and thus associates with the backbone of DNA with high affinity (Figure 5.2) (101,102). This unique binding, “phosphate clamps”, introduced a new DNA binding mode mimicking that of polyarginine, extending the diversity of binding possibilities of platinum chemotherapeutics

(Figure 5.3).

Figure 5.3. Arginine fork (left) and phosphate clamp (right).

DNA Condensing Effects of PPC-NC

DNA has high anionic character due to the negatively charged phosphate backbone. Due to this reason, agents that bind through non-covalent interactions are often cationic. Recently, studies on DNA condensation have received additional attention with considerable potential interest for gene therapy, where the mechanistic details of DNA packing are essential for proper function in the gene regulation processes in living systems (167,168). Moreover, it was found that

70 the cytotoxicity of synthetic polyamine analogues is generally correlated to their ability to condense DNA (169).

Figure 5.4. Pictorial representation showing the mechanism of DNA condensation by cationic molecules. Modified from ref (170).

Condensation of DNA can be induced by different agents: polyamines, multivalent cations, histones, and positively charged polypeptides (171,172,173,174,175,176). Binding of DNA with cationic molecules can be generally described as a three-stage process: initial binding, DNA condensation, and precipitation. Figure 5.4 shows a general schematic of this occurrence by cationic molecules (170). At a low concentration of a cationic molecule, DNA repels each other due to electrostatic repulsion caused by negatively charged phosphate backbone. As the concentration of the cationic molecule increases, the charge on the phosphate backbone is neutralized and electrostatic repulsion is reduced. Then interhelical cross-links occur which, in turn, cause the condensation of DNA (170). Any further increase in the concentration of cationic molecules would result in the neutralization of negative charged of the DNA, which ultimately leads to the precipitation of DNA-cation adduct.

Figure 5.5. Representative AFM images of linearized negatively supercoiled plasmid DNA, pSP73, in the presence of PPC-NC. From Ref (177).

Recently, the DNA condensing effect was examined with PPC-NC by Brabec et al through biochemical and biophysical methods including visualization by atomic force microscopy as shown in Figure 5.5 (177). PPC-NCs were found to condense DNA at ~20 fold lower concentration than spermine, a known DNA condensing cationic molecule. Although biological

72 consequences of DNA condensation induced by PPC-NC is still unclear, it shows another unique feature of PPC-NC resulted, which may be closely related to their unique cytotoxic activities.

The formation of DNA-Pt adducts and perturbation of DNA structure are crucial determinants of the cytotoxicity profile of the compounds. In this section, biophysical studies were utilized to measure the DNA binding affinity and to further observe the condensing effects induced by a series of PPC-NC with varied structures: Monoplatin NC (4+), Diplatin NC (6+), Triplatin

NC (8+), Tetraplatin NC (10+), and AH44 (6+) (Figure 5.1).

5.1.2 Experimental Conditions

PPC-NC were synthesized as reported in Chapter 4. AH44 was synthesized by Ralph Kipping. All other chemicals were obtained from common chemical vendors.

Isothermal Titration Calorimetry

The experiments were carried out at 25°C using a Microcal VP-ITC (Microcal, Northampton, MA).

Calf thymus DNA and drug solutions each were prepared in 10 mM NaClO4 with pH 7.0 with a concentration of 500 μM. All samples were degassed prior to analysis. The platinum solution was titrated into the DNA mixture by making 38 injections of 7.5 μL every 300 sec at a stirring rate of

310 rpm. The actual heats of binding were obtained after subtracting the heats of dilution for the injections of platinum drug into buffer. Origin 7.0 software was used to fit the corrected data and determine the binding affinity (K), changes in enthalpy (ΔH), binding stoichiometry (n), and changes in entropy (ΔS). From these initial measurements, the Gibbs free energy (ΔG) was determined by using Gibbs energy isotherm equation. The molar ratio where precipitation occurs

73 was also obtained by analyzing the curve, which was verified by visual inspection of the sample after the ITC analysis. The experiments were performed in duplicate.

Circular Dichroism

Calf thymus DNA was incubated with the platinum compounds at 37°C in the dark for 1h at following Ri (platinum compound/DNA ratio) values: 0, 0.025, 0.05, 0.075, and 0.1. CD spectra were recorded at room temperature using a Jasco J600 Spectropolarimeter and a 10 mm sub-micro cuvette. The buffer background was subtracted from the obtained spectra. Concentration of DNA was 100 μM bases in a perchlorate buffer (10 mM NaClO4, pH 7.0).

5.1.3 Results and Discussion

Isothermal Titration Calorimetry

Isothermal titration calorimetry (ITC) is a physical technique used to determine the thermodynamic parameters of interactions in solution and has become a widely used method in recent years to study the binding of small molecules to large biomolecules of interest. By using this technique, binding affinity (K), changes in enthalpy (∆H), and binding stoichiometry (n) can be measured, where changes in Gibbs energy (∆G) and changes in entropy (∆S) can be also obtained by using the following equation (Eq. 1).

∆G = -RTlnK = ∆H - T∆S

Isothermal titration calorimetry (ITC) is a useful method to explore the interaction between

DNA and cationic ligands, where ITC is the only technique that can simultaneously determine all binding parameters in a single experiment without requiring modification of binding target.

(178,179). By titrating a solution of cationic molecules into a DNA solution, the thermodynamic parameters such as changes in enthalpy, entropy, and free energy can be obtained by fitting the best ITC curve. Nevertheless, this can be a useful technique to observe a conformational change in DNA, where the phosphate backbone is interacting with PPC-NC thereby causing the DNA to compact, ultimately inducing DNA condensation. Based on the results with previously studied

PPC, cationic PPC exhibited strong DNA binding properties through noncovalent interactions inducing DNA condensation at much lower concentrations than naturally occurring cationic ligands such as spermine and spermidine (177,180). To explore this unique feature and strong

DNA-binding affinity exhibited by PPC-NC in more detail, PPC-NC with different length, charge, and number of platinum centers were studied by ITC.

Figure 5.6. ITC profile for the binding of Triplatin NC with CT-DNA. The isotherm is divided into 3 stages: 1) 1st binding stage 2) condensation 3) precipitation.

Figure 5.7. ITC profiles for the binding of PPC-NC to CT-DNA. A) Monoplatin NC(4+), B) Diplatin NC(6+), C) Triplatin NC(8+), D) Tetraplatin NC(10+), E) AH44(6+).

Table 5.1. Best fit parameters for titration of CT-DNA with platinum compounds. These values were obtained by plotting the best fit on the initial binding stage of the compounds with CT-DNA by using Origin 7.0. a) From ref (181). N.R= Not reported.

In summary, the binding of the platinum compounds with DNA is a thermodynamically favorable event (∆G = negative) where it is driven entropically opposed by unfavorable enthalpy change and favorable entropy change, which is in an agreement with the most of noncovalently binding compounds (Table 5.1). It is known that binding of cations to phosphates on the polynucleotide backbone of DNA is entropically driven by the release of counter-ions and water and opposed by the binding enthalpy (182). The Gibbs free energy (∆G) values were all in the similar range for the compounds studied, where these values were generally lower than the naturally occurring cationic spermidine indicating that these events were more favorable for the

PPC-NC.

The titration curve with CT-DNA consists of three endothermic binding stages (initial binding, condensation, and precipitation), which were labeled and shown in Figure 5.6. The isotherm of the compounds allows for determination of the molar ratios of different binding stages through the visualization of the isotherms. In general, the occurrence of DNA condensation and precipitation occurred faster for the higher charged compounds. The binding stoichiometry (N) for

77 the initial stage showed a similar trend, where this value generally decreased as the charges of the

PPC-NC increased (Table 5.1); however, the condensation process started faster with AH44 (6+) compared to Diplatin NC (6+). Nevertheless, the stoichiometry of AH44 was higher than Diplatin

NC although they were both 6+ charged. The DNA binding profile of AH44 was similar to that of

Triplatin NC (8+), which may suggest that that the number of platinum centers are more crucial in determining DNA binding affinity and the DNA condensing effects of the compounds than the charge state.

Due to the complexity of DNA and the occurrence of multiple events with cationic molecules, many values obtained from these isotherms are generally rough estimates, which heavily depend on the best curve fitting method. From literature, utilizing different plotting techniques has resulted in different binding constant values which differ up to 100 fold on an isotherm of cationic molecules interacting with DNA (183). This discrepancy will influence the the entropy (∆S) and Gibbs free energy (∆G) values obtained from the isotherms, since these values were obtained by using an equation involving the binding constant (K); Therefore, the values obtained from ITC possess a certain degree of inaccuracy and are not further discussed.

Circular Dichroism

Circular dichroism (CD) measures a difference in absorbance by a substance of right- and left- handed circularly polarized light (184,185). An increased relative absorption of left-handed polarized (LPL) light results in a positive band in an absorption spectrum while an increased relative absorption of right-handed polarized light (RPL) will result in a negative band (Eq 2).

Circular dichroism = ΔA(λ) = A(λ)LPL ‐ A(λ)RPL, λ= wavelength

CD is an excellent method for rapidly evaluating the secondary and tertiary structure, and binding properties of biologically-relevant molecules like proteins and DNA at low concentrations.

The typical CD spectrum of a DNA sample is observed in the ultraviolet region between 300 nm and 180 nm, where nucleobases absorb light (186). Changes of bands in the CD spectra allow for distinction of different forms of DNA (B-form, A-form, and Z-form) and monitoring of conformational polymorphism of DNA and DNA-drug complexes (187,188).

Figure 5.8. CD spectra of 3 mM of CT-DNA at various concentration of hexamminecobalt (III). (1) Ri

= 0; (2) Ri =0.098;(3) Ri =0.197; (4) Ri =0.216; (5) Ri =0.256; (6) Ri =0.275; (7) Ri =0.295; (8) Ri =0.314; (9)

Ri =0.334; (10) Ri =0.353. From ref (189).

Change in the secondary structure of DNA through interactions with PPC-NC were observed by CD to confirm the data obtained from ITC and to further investigate (190). Previously,

3+ Bloomfield et al have investigated the interaction of heamminecobalt(III), Co(NH3)6 , with calf thymus DNA (CT-DNA), which revealed two stages of the interaction: binding prior to condensation (Figure 5.8; 1-5) and binding during condensation (Figure 5.8; 6-10) (189). From the ITC study, DNA condensation occurred faster with PPC-NC with higher number of platinum

79 centers and this trend can be confirmed and further explored by utilizing CD to establish the effect of PPC-NCs structural variation on the DNA binding and DNA condensing effects.

Figure 5.9. CD spectra of CT-DNA treated with various amounts of platinum compounds. 100 µM of CT-DNA prepared in 10 mM NaClO4 were treated with platinum compounds at different Ri (DNA to drug ratio) and incubated for 1 h.

Figure 5.10. Changes of the ellipticity at 210 nm (top) and 274 nm (bottom) upon treatment with different Ri (DNA to platinum compounds).

Figure 5.11. Comparison of the compounds at Ri= 0.05 (Top) and Ri=0.1 (Bottom).

Figure 5.9 shows the outcome of the CD experiments of the PPC-NC with CT-DNA.

Increase in the number of platinum center of PPC-NC correlated with their ability to induce conformational changes of CT-DNA, whereas Tetraplatin NC induced the most structural perturbation while Monoplatin NC has induced the least. This effect can be measured by observing the positive band at 274 nm and the negative band at 210 nm of CT-DNA. The positive band at

274 nm initially decreased and the negative band at 210 nm increased as the concentration of the compound increased. At higher concentrations, a complete inversion of the band occurred at 274 nm which was observed with Triplatin NC, Tetraplatin NC, and AH44 which may be indicative of

DNA condensation. This event occurred at a lower Ri (drug to nucleotide ratio) with Tetraplatin

NC compared to the other compounds, indicating a greater condensing effect and DNA binding affinity due to the higher charge and length. The conformational changes occurring at 210 nm and

274 nm were graphed in Figure 5.10, where it showed a general trend that PPC-NC with a higher number of platinum centers have induced greater conformational changes of CT-DNA.

Figure 5.12. Structures of Diplatin NC, AH44, and Triplatin NC

It is also interesting to compare and contrast between Diplatin NC, Triplatin NC and AH44.

Diplatin NC and Triplatin NC utilize dangling amine inert ligand while AH44 utilizes ammine inert ligand. Although Diplatin NC and AH44 have the same overall charge (6+), they showed different DNA binding profiles. They exhibited similar degree of DNA conformational change up to a ratio of 0.05; however, at a ratio of 0.075 and above, AH44 induced dramatic changes in the

CD signal indicating DNA condensation, similar to that of Triplatin NC, which was not observed with Diplatin-NC (Figure 5.11). These results are in good agreement with those obtained from

ITC, where the overall DNA binding profile of AH44 was similar to that of Triplatin NC, while

Diplatin NC induced less DNA structural perturbation. These results suggest that the number of platinum centers in PPC-NC are more crucial determinant in terms of DNA binding than the charge state of the PPC-NC.

5.2 Biological Activities of Polynuclear NC Compounds

5.2.1 Experimental

Cell Culture

HCT116 cells were obtained from the American Type Culture Collection. HCT116 cells were grown in monolayer culture in RPMI 1640 (Invitrogen, Grand Island, NY, USA; Cat# 11875-093) supplemented with 10% fetal bovine serum (Quality Biologicals, Gaithersburg, MD, USA; Cat#

110-001-101US) and 100 U/mL penicillin and 100 mg/mL streptomycin (Invitrogen, Grand Island,

NY, USA; Cat# 15140), at 37 °C in a 5% CO2 incubator.

Cellular Accumulation

HCT116 cells were seeded in 100 mm dishes (2 × 106 cells/plate). After 24 h, cells were treated with 10 µM of the platinum compounds. After 3 h and 6 h, cells were harvested and washed twice with PBS. To digest samples, 1 mL of concentrated HNO3 was added to the pellet and left to digest for three days. 2 mL of water were then added to dilute the samples and the solutions were filtered through a 0.45 μGHP filter, and analyzed on inductively coupled plasma mass spectrometry (ICP-

MS) to determine the platinum concentration. Error bars indicate Mean ± S.D. from two independent experiments. Standards and blanks were prepared the same as the samples.

Growth Inhibition Assay

HCT116 cells were seeded in 96-well plates (5 × 103 cells/well) in 100 μL of RPMi media with

10 % v/v fetal bovine serum, 100 U/ml penicillin, and 100 μg/mL streptomycin. They were allowed to attach overnight. After 24 h, cells were treated with various concentrations (0.78 – 50 μM) of platinum drugs in sets containing 4 replicates. After drug exposure for 24 hours, 1 mM MTT (3-

(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma), was added to each well and incubated for 4 hours at 37 °C. The MTT reagent was removed, and 100 μL of DMSO was added to each well. Spectrophotometric readings were determined at 492 nm using a microplate reader (Bio-Tek instruments).

5.2.2 Cellular Accumulation of PPC-NC in HC116 Cells

Figure 5.13. Cellular accumulation (attomole of Pt/cell) of PPC-NC and AH44 in HCT116 cells.

Previously, Triplatin NC (8+) and AH44 (6+) showed higher cellular accumulation than its covalently binding congener BBR3464 (4+) and cisplatin. To extend the spectrum of the structure-activity relationship exhibited by highly-charged cationic PPC, cellular accumulation of

Mono-, Di-, Tri-, and Tetraplatin NC were assessed in HCT116 (human colon carcinoma) cells after 3 h and 6 h of drug treatment and they were quantified by ICP-MS. Figure 5.13 shows the pattern that platinum levels (attomole of Pt / Cell) were highest with Tetraplatin NC while

Monoplatin NC was lowest as expected; however, Tetraplatin NC has four platinum centers per molecule while Monoplatin NC has one platinum center per molecule. To normalize this, the

86 values were divided by the number of the platinum centers (attomole of drug/ cell) as shown in

Figure 5.14.

Figure 5.14. Cellular accumulation (attomole of drug/cell) of PPC-NC and AH44 in HCT116 cells.

When expressed in attomole drug per cell, Monoplatin NC (4+) displayed the highest amount of drug level in the cells. Similar amount of drug accumulation was observed with Di-,

Tri-, and Tetraplatin NC, while AH44 has shown significantly lower accumulation than those with dangling amine (H2N(CH2)6NH3). This may contradict with our previous conclusion, where it was initially speculated that the higher charge was responsible for the higher accumulation in cells and this correlation was established with BBR3464 (4+), AH44 (6+), and Triplatin NC (8+) (103,156); however, this result suggests that the choice of the inert ligands are a more crucial determinant

87 contributing to cellular accumulation of PPC-NC, whereas PPC-NC with dangling amine linker

(Mono-, Di-, Tri-, Tetraplatin NC) showed higher uptake than that of ammine (AH44).

5.2.3 Growth Inhibition Effects of PPC-NC

Figure 5.15. Cytotoxicity of PPC-NC determined by MTT assay.

Table 5.2. Cytotoxicity of PPC- NC compounds determined by MTT assay in HCT116 cells.

Cytotoxicity of PPC-NC compounds in HCT116 cells was assessed by MTT assay. The results were shown in Table 5.2. Tetraplatin NC was found to be most cytotoxic by exhibiting an

IC50 value of 2 µM, whereas Monoplatin NC did not induce significant cytotoxic activities (>50

µM). This result established a trend that PPC-NC with higher charge and number of platinum centers exhibit higher cytotoxic activities. From the biophysical and cellular accumulation studies,

AH44 showed superior DNA binding profile (Section 5.1) while Diplatin NC resulted in higher cellular accumulation (Section 5.2). Despite the differences in the DNA binding profile and cellular accumulation profiles, they exhibited similar cytotoxic activities in HCT116 cells.

Cytotoxicity exhibited by platinum compounds can be correlated by the frequency and nature of

DNA adducts and the efficiency of cellular platinum accumulation, and this result is thought to be a consequence of combination of these properties of each platinum compound. Overall, we propose Tetraplatin NC as a lead compound, where it demonstrated highest DNA binding affinity and cellular accumulation, which ultimately resulted in highest cytotoxicity.

5.3 Interactions of PPC and Heparan Sulfate

One distinct cellular profiles of PPC-NC is that the cellular uptake is significantly enhanced compared to cisplatin despite of their charge states. The cellular uptake mechanism of neutrally charged cisplatin is known to be mediated by active transporters (e.g. CTR1) or by passive diffusion (139,191). The exact mechanisms of cationic PPC enter cells has not been established, but studies suggest the uptake is mediated by heparan sulfate proteoglycans (HSPG) (156).

Figure 5.16. Pathway for the cationic PTDs (R9) into cells. From ref (192)

Figure 5.17. Major and minor repeating disaccharide sequences of heparin. From ref (193).

HSPG, a type of glycosaminoglycan (GAG), are glycoproteins with the common characteristic of containing one or more covalently attached heparan sulfate chains (194,195,193).

GAGs, including heparan sulfate (HS), heparin, and chondroitin sulfate (CS) are composed of repeating disaccharide units differing in number and position of sulfate groups (196). HSPG are known to mediate the cellular internalization of cationic protein transduction domains (PTDs) and polyamines (192,197,198). Figure 5.16 shows the pathways of cellular internalization of R9, the most efficacious known PTD. There are four steps in the process: (1) binding to cell surface HS,

(2) uptake by endocytosis, (3) release upon HS degradation, and (4) leakage from endocytic vesicles (192). The uptake pathway cationic PPC-NC is suggested to be similar and this was confirmed by cellular accumulation of PPC-NC in wt CHO (chinese hamster ovary), mutant CHO- pgsD-677 (lacking HS), and CHO-475 (lacking HS/CS) cells (Figure 5.18). The cellular accumulation of PPC-NC in all three CHO cells showed GAG dependency, which suggests that

HSPG-mediated interactions are crucial for the cellular internalization of PPC-NC (156).

Figure 5.18. GAG dependent cellular accumulation of PPC-NC. 2 x 106 cells were treated with 10 µM platinum compounds. After 1 h, cells were harvested and washed with PBS buffer. After acid digestion, platinum analysis was performed on ICP-OES. From ref (156).

The interaction of PPC-NC and DNA revealed that PPC-NC binds to negatively charged phosphate backbone of DNA similar to that of cationic arginine, where this new mode of binding is described as “phosphate clamp”. Similarly, the interaction of PPC-NC and HS is speculated to be a consequence of positively charged PPC-NC and negatively charged sulfates on HS, where this “sulfate clamp” motif is suggested and shown in Figure 5.19.

Figure 5.19. Arginine fork, phosphate clamp, and sulfate clamp.

5.3.1 Cellular Accumulation of PPC-NC in HSPG deficient Cells

Experimental Conditions

CHO wt and CHO-745 cells were seeded in 100 mm dishes (2 × 106 cells/plate). After 24 h, cells were treated with 10 µM of the platinum compounds. After 3 h, cells were harvested and washed twice with PBS. To digest samples, 1 mL of concentrated HNO3 was added to the pellet and left to digest for three days. 2 mL of water were then added to dilute the samples and the solutions were filtered through a 0.45 μGHP filter, and analyzed on ICP-MS to determine the platinum concentration. Error bars indicate Mean ± S.D. from two independent experiments. Standards and blanks were prepared the same as the samples.

Results

Figure 5.20. Cellular accumulation of PPC-NC compounds in CHO wt and CHO-745 HSPG deficient cells. Cells were treated with 10 µM of platinum compounds for 3 h.

The intracellular platinum concentration of PPC-NC in CHO wt and CHO-745 (deficient in HS and CS) was measured to confirm the dependency of PPC-NC compounds on HSPG status of the CHO cells. The result confirmed that cellular internalization of PPC-NC heavily depends on the status of HSPG by showing significantly lower cellular accumulation in HSPG deficient cells, while cisplatin did not show any dependency on HSPG status. Previously, it was suggested that the HSPG-mediated internalization correlates with the charge state of the compounds (156); however, as shown in Figure 5.20, the cellular accumulation of PPC-NC in CHO wt and CHO-745 was dependent on the choice of the inert ligand rather than the charge of the compound, which confirmed the result of cellular accumulation study in HCT116 cells (Section 5.2).

5.3.2 Interaction with D-glucosamine-3-sulfate by HSQC [1H, 15N] Spectroscopy and

ITC

Figure 5.21. Structure of 15N labelled BBR3464.

Experimental Conditions

Starting Materials

D-glucosamine-3-sulfate was purchased from Sigma Aldrich. 15N-labelled BBR3464 was prepared with 15N labelled ammine and 1,6-hexanediamine by using a method described in Section 4.2.1.

2D [1H, 15N] HSQC NMR Spectroscopy

The NMR spectra were recorded on a Bruker AVANCE 600 MHz spectrometer (1H, 599.92 MHz;

15N, 60.79 MHz). The 1H spectra were internally referenced to 1,4 dioxane (δ= 3.767 ppm), and

15 15 the N chemical shifts were externally referenced to NH4Cl (1.0 M in 1.0 M HCl in 95% H2O/5%

15 1 D2O) at δ( N)=0.0 ppm. The H NMR spectra were acquired with water suppression using the

WATERGATE pulse sequence (199). The 2D [1H, 15N] HSQC NMR spectra (decoupled by irradiation with the GARP-1 sequence during acquisition) optimized for 1J(15N,1H)=72 Hz were recorded using the standard Bruker phase sensitive HSQC pulse sequence (200). One-dimensional

1H spectra were recorded with 32 transients, a spectral width of 12 kHz, and a relaxation delay of

1.5 s. For the 2D [1H, 15N] HSQC NMR spectra, 8 transients were collected for 128 increments of

1 15 t1 with an acquisition time 0.152 s and spectral widths of 2 kHz in ƒ2 ( H) and 1.8 kHz in ƒ 1 ( N).

The spectra were processed using Gaussian weighting functions in both dimensions.

Isothermal Titration Calorimetry

Procedure was followed as described in Section 5.1.2.

Results

Figure 5.22. Structure of glucosamine-3-sulfate.

Heparin sulfate is extremely difficult to analyze and quantify since they are polydiverse and microheterogeneous polymers where the molecular weight ranges from 3 kDa to 30 kDa (201).

To overcome this obstacle, 15N labelled PPC was synthesized to allow for the detection and quantification of the interaction by HSQC [1H, 15N] NMR spectroscopy. Initially, the interaction of PPC-NC and heparin sulfate was attempted; however, the PPC-NC and heparin sulfate adducts could not be characterized due to an immediate precipitation of the final product. A less complex molecule, glucosamine-3-sulfate (G3S), a monosaccharide unit of HS, was chosen in this study as a probe to observe an interaction between PPC-NC and heparin sulfate. The interaction of G3S and BBR3464 was observed up to 72 h; however, no changes were observed in the spectra as

96 shown in Figure 5.23, suggesting that the interaction between sulfated monosaccharide G3S was not strong enough to be detected.

Figure 5.23. HSQC [1H, 15N] of 2 mM of BBR3464 with 8 mM glucosamine-3-sulfate (G3S). A) BBR3464 without G3S. B) BBR3464 with G3S after 72 h. NH3 region: (I) PtN3Cl, (II) PtN4. NH2 region: (III) PtN4.

PtN3Cl in NH2 region is not shown in this spectra since it overlaps with the water peak.

To confirm this result, isothermal titration calorimetry was utilized to detect interactions of G3S with BBR3464 and Triplatin NC since it can detect the interaction between two molecules by measuring the change of heat released or absorbed with high sensitivity. Based on the isotherm, it can be concluded that the interaction of PPC and G3S was not significant enough to detect

(Figure 5.24). This may suggest that individual noncovalent bond between monosaccharide and

PPC are individually weak; but multiple noncovalent interactions allow to form strong adduct between HS and PPC.

Figure 5.24. Isothermal Titration Calorimetry of BBR3464 and G3S.

5.3.3 Colorimetric Assay to Quantify PPC-NC and Heparin Sulfate Interaction

Figure 5.25. Structure of methylene blue (MB) dye.

A monosaccharide unit of heparin sulfate, G3S, did not show any interaction with PPC-

NC; therefore, there was a need to develop a method to detect between PPC-NC and heparin sulfate.

To accomplish this, a colorimetric assay using methylene blue (MB) was developed. MB is a cationic dye which is known to interact with negatively-charged functional groups on polysaccharides through electrostatic interactions. This interaction causes a shift in the absorption spectrum of MB, which can be used for the detection and quantification of heparin (202). Cationic

PPC-NC binds to heparin strongly and the pre-treatment PPC-NC prevented the binding of MB to heparin which allowed the quantification of PPC-NC and heparin adduct.

Experimental

Heparin sulfate was purchased from Sigma Aldrich. Proteoglycan detection kit including methylene blue dye was purchased from Astarte Biologics (Cat #8000).

Preparation of standards and samples

Stock standard solutions were prepared in deionized water on the day of analysis. Standards were prepared in the concentration of 10, 7.5, 5, 2.5, and 1.25 µg/mL of heparin sulfate solution.

Samples were prepared by adding 10 µL of 0.1 mM platinum compounds to 200 µL of 10 µg/mL in a test tube. The samples were allowed to react for 30 mins prior to the analysis. The samples were prepared in duplicates.

UV-Visible Spectroscopy

Standards and the samples were measured by using HP (Agilent) 8453 UV/Vis spectroscopy. To

150 µL of the standards or samples, 150 µL of methylene blue was added and vortexed, then

99 subsequently measured by UV-Vis spectroscopy. Full visible absorption spectrum (400-800 nm) was measured for all of the standards and samples to determine the optimal wavelength for the quantification.

Determination of Calibration Curve

Figure 5.26. Absorption spectra of standards. To 150 µL of heparin samples at various concentrations (0 µg/mL to 10 µg/mL), 150 µL of MB solution was added, then measured immediately.

Figure 5.26 shows the absorption spectra MB and MB-heparin complexes from 400 nm to

800 nm. With increased heparin concentration in the mixture, the absorption maxima of MB at

650 nm and 595 nm decreased, while a new absorption (heparin-MB) peak at 525 nm increased.

Previous published methods on heparin detection utilized the absorbance at 525 nm to observe the interaction of methylene blue and sulfated molecules, where a new absorption peak appears

(203,202); however, based on the full spectra generated by various concentrations of standards, 100 measuring a decrease in absorbance peaks at 650 nm provided better linearity and higher sensitivity than measuring the absorbance at 525 nm (Figure 5.26).

Figure 5.27. Calibration curve at 650 nm.

This was also confirmed with the absorption spectra obtained upon PPC-NC treatment

(Figure 5.28). Absorption intensity of the peaks at 525 nm did not change significantly while absorption at 595 nm and 650 nm demonstrated higher sensitivity. Overall, 650 nm provided the highest sensitivity with best linearity with R-squared value of 0.9977 (Figure 5.27) and this wavelength was selected to study the interaction of heparin and PPC-NC.

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Figure 5.28. Full absorption spectra upon treatment with PPC-NC. 150 µL of 10 µg/mL of heparin sulfate solution was treated with 2.4 µM of PPC-NC. To this mixture, 150 µL of MB solution was added then subsequently measured by UV/Vis spectroscopy.

Figure 5.29. Absorbance intensity at 650 nm after treatment with platinum compounds. 150 µL of 10 µg/mL of heparin sulfate solution was treated with 2.4 µM of PPC-NC. To this mixture, 150 µL of MB solution was added then subsequently measured by UV/Vis spectroscopy.

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As shown in

Figure 5.29, Tetraplatin NC inhibited MB-heparin interaction most effectively compared to other platinum compounds examined. The inhibition of MB-sulfate interaction by platinum compounds can be linearly correlated by charge of the PPC-NC, where an increase in charge and number of platinum center resulted in less heparin-MB binding. Interestingly, AH44 (6+) interacted with heparin more effectively than Diplatin NC (6+) and exhibited a similar heparin binding profile with Triplatin NC. This result is in an agreement with PPC-NC binding with DNA, where the binding profile of AH44 was similar to that of Triplatin NC. Based on these findings, it is suggested that number of platinum center is more crucial than charge of the PPC-NC in terms of binding with heparin.

5.3.4 Effects of Heparin in DNA-Pt Binding

Experimental

CD experiments were performed as reported in Section 5.1.2. As stock solutions used in this study,

1 mM of DNA solution, 100 µM of Triplatin NC solution, and 300 µg/mL of heparin sulfate solution were prepared in 10 mM of NaClO4. Further dilutions were made with 10 mM of NaClO4 to achieve desired final concentrations. Incubation was done in 37° C to mimic physiological conditions. Experiments were performed in triplicate to confirm the results.

Results and Discussion

The mechanism of the cytotoxicity exhibited by PPC-NC is the PPC-NC and DNA adduct formation through noncovalent interactions, which will cause structural disturbance of DNA, ultimately triggering apoptosis. Previous studies have suggested that the uptake mechanism of

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PPC-NC is mediated by HSPG, where it enters the cells through endocytosis. To exhibit cytotoxicity, an internalized PPC-NC has to be released from the heparin sulfate in order to interact with DNA to cause a structural perturbation. To investigate this, PPC-NC was allowed to react with heparin sulfate before and after incubation with DNA and this interaction was monitored by

CD.

Figure 5.30. Effects of Heparin Sulfate on DNA Binding. A) 100 µM of CT-DNA was incubated with 30 µg/mL heparin sulfate for 1 h. B) 100 µM of CT-DNA was incubated with 10 µM of Triplatin NC for 1 h. C) 10 µM of Triplatin NC was incubated with 60 µg/mL heparin sulfate for 1 h, then 100 µM of CT-DNA was added and incubated for additional 1 h. D) 100 µM of CT-DNA was incubated with 10 µM of Triplatin NC for 1 h, then 60 µg/mL heparin sulfate was added and incubated for additional 1 h.

Interestingly, there were no observed conformational changes in CT-DNA when PPC-NC was incubated with heparin prior to the interaction with CT-DNA (Figure 5.30 C). This interaction

104 was followed for several days to observe the reversibility of Pt-heparin adduct; however, no conformation change was observed even after 96 h. This may suggest that the Pt-heparin adduct is not easily reversible or PPC-NC preferentially bind to heparin over CT-DNA. Another experiment was performed to test this hypothesis, where PPC-NC was incubated with CT-DNA for 1 h, followed by incubation with heparin for addition 1 h (Figure 5.30 D). Even in this case, no CT-DNA conformation change was observed by CD indicating that Pt-DNA adduct has been reversed. Based on this finding, PPC-NC preferentiality binds to heparin versus CT-DNA. Further investigation is necessary to examine this phenomenon.

Figure 5.31. Effects of heparinase I on PPC NC and heparin adduct. A) 100 µM of CT-DNA B) 10 U/mL of heparinase I C) 100 µM of CT-DNA was incubated with 10 µM of Triplatin NC for 1 h, then 60 µg/mL heparin sulfate was added and incubated for additional 1 h. C) 60 µg/mL heparin sulfate was incubated with 10 µM of Triplatin NC for 1 h, then 100 µM of CT-DNA was added and incubated for additional 1 h. D) 30 µg/mL heparin sulfate was incubated with 10 µM of Triplatin NC for 1 h, then 100 µM of CT-DNA was added and incubated for additional 1 h. To the mixture, heparinase I was added and incubated for 0.5 h.

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Based on the CD study of heparin and Triplatin NC, Triplatin NC preferentially binds to heparin, whereas it needs to be released from the heparin to exhibit cytotoxic activity by interacting with DNA. To explore a possible mechanism of its release from heparin, heparinase I was added to the solution, where Pt-heparin adducts were present. Heparanase I is an enzyme known to cleave hepaain and is essential for the release of polyamines (e.g. spermine, spermidine) after cellular internalization mediated by heparan sulfate proteoglycans (HSPG) (192,204,205).

The CD spectrum of DNA shows a distinct positive band at 274 nm, whereas heparinase I does not contribute any CD signal at this wavelength. When CT-DNA was treated with Triplatin

NC pre-incubated with heparin, no DNA conformation change was observed (Figure 5.31 C); however, when heparinase I was added to the mixture, the positive band at 274 nm from CT-DNA became a negative band, indicating a significant conformation changes of CT-DNA structure

(Figure 5.31 C). This may suggest that the mechanism of PPC-NC is similar to cell entry of cationic polyamine mediate by HSPG, where PPC-NC binds to cell surface HSPG, uptake by endocytosis, release upon heparan degradation through heparanase, then ultimately binding to

DNA. Although PPC-NC showed to inhibit the enzymatic activity of heparanase in previous study, it is suggested from this result that heparanese may be responsible for the release of PPC-NC bound to HSPG in the cells. Yet, one study is not enough to conclude the complex cellular entry mechanism of PPN-NC, and further investigation are necessary to confirm these findings and to fully understand the mechanism of cellular internalization of PPC-NC.

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5.4 Contributions

Cytotoxicity was done by Samantha Katner.

Daniel E. Lee and Samantha Katner contributed equally to cellular accumulation study.

All of other work reported in this chapter was done by Daniel E. Lee.

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Chapter 6

Other Contributions

108

CHAPTER 6. OTHER CONTRIBUTIONS

6.1 Hybrid Polynuclear Platinum Compounds

A major problem of BBR3464 was degradation of the compound into inactive metabolites, where one of the fragments shown in blood metabolism studies was identified as Monoplatin NC

(206). This pharmacokinetic problem has discouraged to continue the investigation of BBR3464 in the clinical setting. A cell proliferation study with PPC-NC reported in section 5.2 shows that

Monoplatin NC did not exhibit significant cytotoxic activity in cells, while Di-, Tri-, and

Tetraplatin NC exhibited micromolar cytotoxic activity in cells. This gave a motivation to further investigate the PPC-Cl, where one of the fragments of Tetraplatin-Cl and Pentaplatin-Cl formed upon inactivation by sulfur molecules are cytotoxic Diplatin NC and Triplatin NC, respectively

(Figure 6.1).

Tetraplatin-Cl and Pentaplatin-Cl are predicted to exhibit two distinct modes of action: 1) exhibits nanomolar cytotoxicity as seen with BBR3464 by covalently binding with DNA. 2) if they are degraded by sulfur nucleophiles, they will release Diplatin NC and Triplatin NC to exhibit micromolar cytotoxicity by interacting with DNA through noncovalent interactions. This novel approach may trigger two distinct cell death mechanism by exhibiting different modes of DNA binding and which may resolve the pharmacokinetic problems of BBR3464. To allow investigation of this hypothesis, Tetraplatin-Cl(synthesized in Chapter 4) and Pentaplatin-Cl are synthesized, characterized, and evaluated.

109

Figure 6.1. Metabolites of PPC-Cl analogs produced upon metabolic degradation by sulfur nucleophiles. Tri-, Tetra-, and Pentaplatin-Cl releases Mono-, Di-, and Triplatin NC, respectively.

110

6.1.1 Synthesis of Pentaplatin-Cl

Figure 6.2. Synthetic scheme of Pentaplatin-Cl

Syntheses and characterization of BBR3464 and Tetraplatin-Cl are described in Section 4.2.

Pentaplatin-Cl

To 1 mmol of Triplatin NC dissolved in DMF, 2.2 mmol of DIPEA was added while stirring. 2.2 mmol of mono-activated transplatin was added to the solution and the mixture was allow react for

2 days at ambient temperature. The solution was evaporated to dryness under reduced pressure and was dissolved in minimum amount of water. The reaction was filtered through syringe filter to remove any unreacted starting material. The filtrate was evaporated to ~1 mL under reduced pressure and 25 mL of acetone was added to precipitate the product. The precipitant was collected through filter and was washed with acetone.

111

1 Pentaplatin-Cl- (Yield: 39%). H NMR (D2O): δ 2.7ppm (16H, m), 1.7(16H, m), 1.4 (16H, m).

Elemental Anal calc’d for C24H94Cl2N26O24Pt5- C 13.24, H 4.35, N 16.72. Obtained- C 13.33, H

4.38, N 16.68.

6.1.2 Active Metabolites of Novel PPC-Cl with Dual Antitumor Activity

Experimental Conditions

Electrospray ionization time of flight mass spectrometry

Platinum compounds were prepared at a final concentration of 3 mM and were incubated with N- acetyl-cysteine in a reaction ratio of 1:4 (platinum complex: N-acetyl-cysteine) in a mixture of water and methanol (70: 30, v:v) for 16 h. pH was adjusted to 7.4 using NaOH. Experiments were conducted on a Waters/Micromass QTOF-2 instrument operated in positive ion mode. An electrospray voltage of 4.5kV and a capillary temperature of 200 ºC were used. The infusion rate was 3 uL/min. Collision gas was introduced into the hexapole to aid in ion cooling. All spectra were analyzed using the MassLynx 4.0 software provided from the manufacturer.

195Pt NMR Spectroscopy

Platinum compounds were prepared at a final concentration of 3 mM and were incubated with N- acetyl-methionine in a reaction ratio of 1:4 (platinum complex: N-acetyl-cysteine) in H2O/D2O

(70:30, v.v) for 16 h. pH was adjusted to 7.4 by using NaOH. 195Pt spectra were referenced to

195 Na2[PtCl6]. The scanning frequency for Pt nuclei was set at 64.32 MHz. All spectra were analyzed using Mestranova 8.1 software.

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Figure 6.3. Proposed structures of PPC-Cl.

Results and Discussion

The structures of the platinum compounds studied are shown in Figure 6.3. ESI-MS and

195Pt NMR spectroscopy were utilized to confirm our hypothesis that PPC-NC will be released upon the metabolic degradation of PPC-Cl. BBR3464, Tetraplatin, and Pentaplatin-Cl were allowed to react with 4 equivalents of n-acetyl-cysteine at pH 7.4 for 16 hours to allow complete degradation. N-acetyl-cysteine was specifically selected for this study since thiols are abundant in a physiological environment (e.g. glutathione) and are known to rapidly degrade platinum compounds into inactive metabolites.

Figure 6.4. Inactive metabolites produced by PPC upon the interaction with NAC.

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Figure 6.5. Reaction of PPC with NAC followed by ESI-MS. i. BBR3464 with NAC. ii. Tetraplatin- Clwith NAC. iii. Pentaplatin-Cl with NAC. A. PPC-NC: Monoplatin NC(i), Diplatin NC(ii), and Triplatin

+ NC (iii). B. Inactive metabolite: [Pt(NH3)2(NAC)2] . C. Thiolate bridged inactive metabolite [(NAC)(NH3)2Pt-

+ NAC-Pt(NAC)(NH3)2] . (The structures of B and C are shown in Figure 6.4).

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Upon the reaction of BBR3464 with NAC, Monoplatin NC (460.27 m/z, 1+) was observed as one of its metabolites (Figure 6.5i). Sulfur bound inactive platinum metabolite B and sulfur- bridged metabolite C were also observed at 554.07 m/z (1+) and 944.10 m/z (1+), respectively

(Figure 6.5). Similar products were seen with Tetraplatin-Cl and Pentaplatin-Cl, where they produced Diplatin NC (268.47 m/z, 4+; 402.21 m/z, 3+) and Triplatin NC (287.39 m/z, 4+; 382.85 m/z), as well as inactive metabolite B and C (Figure 6.5ii-iii). The results from this study confirms our hypothesis by producing PPC-NC compounds as one of their metabolites.

Figure 6.6. Reaction of PPC-Cl with NAC followed by 195Pt NMR spectroscopy. A. Tetraplatin-Cl, B. Tetraplatin-Cl with NAC. 115

To confirm this result, 195Pt NMR spectroscopy was also utilized to identify the metabolites under the same condition (Figure 6.6). 195Pt NMR spectroscopy is a very useful tool to distinguish different ligand substituents bound to the platinum compounds. Upon the reaction with NAC, PPC-

Cl produced PPC-NC (PtN4) and their inactive metabolites (PtN2S2) as shown in Figure 6.6. By utilizing these chemical techniques, the release of PPC-NC upon metabolic degradation of PPC-

Cl was confirmed. These structurally novel compounds with active metabolites can be a solution to the pharmacokinetic problems of BBR3464; furthermore, it can potentially reduce the possibility of developing drug resistance by exhibiting two distinct mode of action. Further investigation including biological assays are deserved to confirm that these compounds exhibit dual antitumor activity in cells.

6.2 Synthesis of a Carboxylate Derivative of BBR3464

The utilization of carboxylate strategy was proven to enhance the pharmacological properties of cisplatin by allowing slower substitution kinetics while it has also resulted in higher cellular accumulation and increased water solubility (207,208,209). Utilizing more stable carboxylate leaving group in place of chloride on TPA compounds also resulted in improved pharmacological profiles including enhanced stability to sulfur nucleophiles (84,71,66). Phase II clinical drug BBR3464 was discouraged due to rapid degradation into inactive metabolites. To address the pharmacokinetics problems of BBR3464, a carboxylate derivative of BBR3464 was synthesized as shown in Figure 6.7.

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Figure 6.7. Synthetic scheme of BBR3464-Acetate.

Synthetic Procedure of BBR3464-Acetate

The synthesis of BBR3464-acetate is adopted and modified from a method published previously (210)

0.5 mmol of BBR3464 chloride salt was dissolved in 10 mL water. 3.0 mmol of silver acetate was added and the mixture allowed to stir overnight in dark at 50°C. Next day, the solution was filtered through membrane filters twice to remove silver chloride formed. Then the solution was evaporated to minimal volume (0.5 ~ 1 mL). Then 20 mL of acetone and ether were added and stirred at 4°C to allow precipitation. The product was collected by filter and then was washed with acetone. If further purification was necessary, the crude product was recrystallized from water.

1H NMR: δ 2.65 (quin, 8H), 2.0 (s, 6H), 1.9 (s, 12H), 1.67 (m, 8H), 1.38(m, 8H). Elemental

Analysis: Expected - C, 22.62, H 5.38, N 10.99. Found- C 22.25, H 5.11, N 10.46.

trans configuration platinum compounds (e.g TPA) utilized weak trans influencing carboxylates in trans position to another, which resulted in significantly enhanced stability than its chloride analog, while cis configuration platinum compounds (oxaliplatin and carboplatin) have utilized bidentate carboxylate to accomplish the enhanced stability. However, BBR3464 has the leaving group trans axial to amine linker, which exerts greater trans influence than the carboxylates, which may result in faster substitution kinetics. As a consequence, BBR3464-acetate

117 did not significantly enhance the stability of the compound; therefore, further investigation of PPC carboxylates was not made.

Although carboxylate strategy on BBR3464 did not result in enhanced stability, this compound may be useful for other purpose. Our collaborator from University of British Columbia

(Center for Drug Research and Development) is currently investigating on liposomal formulation of BBR3464, which is intended to protect the drug against metabolic inactivation, and to deliver more drug more selectively to the site of tumor growth, thereby increasing efficacy and lowering toxicity. BBR3464-acetate may enhance the association with the surface of liposomal nanoparticle, where this will be further investigated by our collaborator.

6.3 Synthesis of TPA Succinate Compound.

The usage of cisplatin is limited due to its toxic side. The lack of specificity of conventional cytotoxic platinum agents is a major clinical problem. Conventional antitumor agents have a narrow therapeutics window and are distributed to normal and cancer cells in nonselective manner

(137). A key objective of this was to develop platinum based drugs with equal cytotoxicity but reduced toxicity by enhancing its specificity to tumor tissue.

Tumor cell survival, growth, and metastasis are driven by angiogenesis, the process by which new blood vessels are formed. Tumor blood vessels are morphologically distinct from normal blood vessels and it has become a major target for treating cancer selectively (211,212). Among the cell surface proteins upregulated in tumor cells during tumor growth and metastasis, αVβ3 and

αVβ5 integrin are attractive targets since they recognize a peptide motif containing RGD (Arg-Gly-

Asp) motifs (Figure 6.8) (213).

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Figure 6.8. Structure of RGD tripeptide

Figure 6.9. Structure of trans-Pt[(isq)(NH3)(succinate)2].

On the basis of these finding, we have attempted to conjugate RGD tripeptide to our TPA compounds to enhance its specificity to target tumors. TPA compounds are suitable for this purpose since it employs leaving carboxylate group trans axial to each other, which significantly slows down the rate of hydrolysis due to low trans effect and influence exerted by the carboxylates.

To allow conjugation of RGD peptide to TPA compounds, the compound shown in Figure 6.9 was

119 synthesized. This structure allows RGD coupling with terminal carboxylate groups of the succinate moieties.

Synthetic Consideration

During the synthesis of succinate platinum compounds, platinum compounds may polymerize since platinum can bind with either carboxyl moiety of succinic acid. Synthesis of trans-[Pt(isq)(NH3)(succinate)2] was initially attempted by utilizing mono-methyl protected succinate, followed by acid hydrolysis, to avoid polymerization of the platinum compound (Figure

6.10). However, this attempt was not successful since isoquinoline ligand and Pt-carboxylate bond were more vulnerable to acid hydrolysis than methyl group on the carboxylate. In an alternate approach, excess amount (20 equivalents) of succinic was added to trans- [Pt(isq)(NH3)(I)2] activated with silver nitrate and this method successfully produced the desired product (Figure

6.11).

6.3.1 Attempt to synthesize the succinate TPA by utilizing methylated succinate

followed by acid hydrolysis

Figure 6.10. Attempt to synthesize trans-Pt[(isq)(NH3)(succinate)2] by using methylated succinate

trans-[Pt(isq)(NH3)(I)2]- Prepared as reported in section 2.2.

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Silver methyl succinate- 2 mmol of mono-methyl succinate was dissolved in 10 ml of water. 1.2 mmol of silver oxide was added and stirred in dark for 16 h. Excess silver oxide was removed by filtering through 0.2µm syringe filter. The filtrate was lyophilized in dark and white powder was

1 collected. H NMR(D2O): δ 3.6(s, 3H), 2.7(m, 4H).

trans-[Pt(isq)(NH3)(mono-methyl succinate)2]- 1 mmol of trans-[Pt(isq)(NH3)I2] was suspended in water. 1.98 eq. of silver methyl succinate dissolved in water was slowly added to the solution.

The reaction was allowed to stir overnight in dark. Next day, silver iodide formed during the reaction was removed by filtering through celite. The filtrate was evaporated to dryness under reduced pressure. The solid was suspended in acetone, filtered, and washed with methanol/acetone.

The product was characterized by 1H NMR spectroscopy and CHN elemental analysis. 1H NMR

(D2O): δ 9.6 (s, 1H), 8.7(d, 1H), 8.3(d, 1H), 8.1(d, 1H), 7.9(d, 1H), 7.6(m, 2H), 3.6(s, 6H), 2.7(m,

8H). Anal. Calc’d for C19H24N2O8Pt: C 37.81, H 4.01, N 4.64. Found: C 37.62, H 4.12, N 4.49.

Acid hydrolysis- Acid hydrolysis was attempted by using following acid at different concentrations, temperature, and reaction time: Hydrochloric acid, nitric acid, trifluoroacetic acid, phosphoric acid, and acetic acid. Based on 1H NMR spectroscopy, it was evident that isoquinoline on TPA compound was more susceptible to acid than the methyl group on the succinate, where the peak corresponds to isoquinoline has disappeared significantly faster than methyl group on succinate. At this point, an alternate approach was investigated.

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6.3.2 Synthesis of TPA-succinato compounds by using excess succinic acid

Figure 6.11. Synthesis of trans-Pt[(isq)(NH3)(succinate)2] by using excess succinic acid.

trans-[Pt(isq)(NH3)(succinate)2]- To 0.5 mmol of trans-[Pt(isq)(NH3)I2] suspended in water, 1.0 mmol of silver nitrate was added. The mixture was stirred for overnight in dark at room temperature. Silver iodide formed was filtered through celite and 10 mmol of succinic acid was added to the filtrate. The mixture was stirred at room temperature for 3 days. The solution was evaporated to dryness and was dissolved in minimum amount of warm methanol. The product was precipitated with ether. Precipitated product collected through filtration and was washed with

1 acetone and ether to remove any residual succinic acid. H NMR ((D2O): δ 9.6 (s, 1H), 8.7(d, 1H),

8.3(d, 1H), 8.1(d, 1H), 7.9(d, 1H), 7.6(m, 2H), 2.6(s, 8H). Anal. Calc’d for C17H20N2O8Pt: C

35.48, H 3.50, N 4.87. Found: C 35.62, H 3.52, N 4.69.

122

Figure 6.12. Conjugation of RGD peptide to TPA.

Next step of this research involves coupling amine group of glycine in RGD peptide to carboxylate terminal end of succinate moiety, which will be prepared as reported by Lippard et al

(Figure 6.12) (214). Next step was not accomplished since the coupling chemistry and separation techniques used in this method were not within our expertise. This work may be re-attempted in future.

6.4 Conjugation of NBD-Fluorophore

Polynuclear platinum compounds (PPC) are a structurally discrete class of platinum compound whose pharmacological properties significantly differ from those of cisplatin. The mechanism of cellular internalization was not thoroughly studied, yet they have shown significantly higher cellular accumulation than cisplatin, while demonstrating micromolar cytotoxicity in tumor cells. The use of molecular imagining techniques, such as confocal microscopy, is an important tool in understanding of the drug trafficking and intracellular distribution. Previous utilization of NBD (7-nitro-2,1,3-benzoxadiazole) fluorophore was made by conjugating to Triplatin NC and cisplatin to examine the effects of protein association on intracellular localization by confocal microscopy (215). The advantage of the NBD fluorophore is

123 that the size of the fluorophore is relatively small compare to other commercially available fluorophores and NBD derivatives of Triplatin NC and cisplatin did not have much influences on the biological properties of the platinum compounds.

The conjugation of NBD fluorophore can be made by attaching to unbound amine of hexamethylenediamine linker of PPC, where the structure of analogues are not significantly different from the parent compounds as shown in Figure 6.15. To further understand the cellular internalization of PPC, an attempt was made to synthesize a series of PPC-NBD analogues, where the compounds differ in charge, length, and the number of platinum centers as shown in Figure

6.13.

Figure 6.13. Proposed Structures of PPC-NBD Compounds

124

6.4.1 Preparation of 6-NBD

Figure 6.14. Preparation of 6-NBD ligand.

All of the chemicals including NBD-Cl and n-boc-1,6-hexanediamine were purchased from common vendors.

Adopted synthetic procedures from (216).

N-(tert-Butoxycarbonyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)hexamethylediamine

A solution of 7-nitrobenzo-2-oxa-1,3-diazol-4-yl chloride (0.398 g, 2.0 mmol) in 3.0 mL of methanol was added dropwise in 5 min., under a nitrogen atmosphere at room temperature, to a stirred solution of the N-BOC-1,6-diaminohexane (2.0 mmol) and triethylamine (0.31 mL, 2.2 mmol) in 4.0 mL of the same solvent. The dark solution was stirred at room temperature with the exclusion of light for 15-20 h. The mixture was diluted with water and extracted with CHCl3. The organic phase was dried over MgSO4 and evaporated to dryness.

Preparation of N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)hexamethylendiamine hydrochloride

(6-NBD)

N-(tert-Butoxycarbonyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)hexamethylediamine(1.9 mmol) was dissolved in 8.0 mL of ethyl acetate and treated with 1.9 mL of 3 M HCl. After being stirred at 60 ºC for 3 h, the mixture was concentrated at reduced pressure and the residue was washed

125

1 with chloroform, yielding pure final product. Yield (72%). H NMR (CD3OH): δ 8.5 (d, 1H), δ

6.33 (d, 1H), δ 3.4 (m, 2H), δ 2.7 (m, 2H), δ 1.6 (m, 2H), δ 1.45 (m, 2H), δ 1.3 (m, 4H).

6.4.2 Attempt to Conjugate 6-NBD to PPC

Figure 6.15. General Scheme of NBD-Conjugation

Synthesis of 6-NBD was initially attempted by a synthetic method described by Chen et al

(217), where they react 1,6-hexanediamine with NBD-Cl. Greatest challenge with this method was the formation of side product (bis-NBD-hexanediamine), where silica gel chromatography was required to remove the side product formed during the reaction. This method was performed in large volume of solvent and cold temperature (0 C°) to minimize the formation of side product; however, the formation of the side product was inevitable with this approach. Thus, further purification was necessary to obtain the desired product, whereas this method was very time consuming with low yields of the products.

Different approach reported by Taliani et al was investigated, where mono-n-boc-1,6- hexanediamine was conjugated to NBD-Cl followed by boc-deprotection. This method dispenses the formation of the bis-NBD product (Figure 6.14) and allowed cleaner, faster, and more efficient synthesis of 6-NBD. This method was utilized throughout this section to obtain 6-NBD required for further syntheses.

126

Conjugation of 6-NBD was attempted by following the synthetic procedure of PPC-NC, where “dangling amine” was conjugated to PPC-Cl (Section 4.3). The starting materials

(transplatin, 1,1/t,t, BBR3464) were synthesized by synthetic procedures reported in Section 4.2.

The general synthetic schematic of NBD-derivative is shown in Figure 6.15, where the platinum compounds were activated by adding 2 equivalents of silver nitrate, then 2.2 equivalents of deprotonated 6-NBD ligand was added to accomplish the desired product.

The initial conjugation of 6-NBD was attempted on transplatin, which is the simplest compound in the series; however, pure product was not obtained. Many purification methods were attempted to purify the final product; washing with various organic solvents (acetone, acetonitrile, and ether), recrystallization (methanol/dichloromethane), soxhlet extraction (dichloromethane), and liquid-liquid extraction (water/dichloromethane). None of these purification steps allowed the isolation of the pure product. This synthesis was also attempted on 1,1/t,t to synthesize Diplatin-

NBD; however, pure product was also not obtained.

Figure 6.16 (bottom) shows the most promising batch of Monoplatin-NBD obtained. A shift of a peak from 2.4 ppm (protons of CH2-NH2) to 2.7 ppm (CH2-NH2-Pt) shows the occurrence of the conjugation; however, there was a discrepancy of a peak integration at 3.5 ppm (CH2-NH-

NBD), where this reduced integration was found. The cause of this occurrence is not known and this was also seen with NMR spectrum of Diplatin-NBD. CHN elemental analysis was obtained to further characterize the compound, whereas obtained CHN values were significantly different from theoretical CHN values, indicating that the obtained product was not pure. (Monoplatin-

NBD: Anal. Calc’d for C24H40N14O12P- C, 31.62; H, 4.42; N, 21.51. Found – C, 36.07; H, 3.92;

N, 20.87, Diplatin-NBD: Anal. Calc’d for C30H62N20O18Pt2- C, 26.09; H, 4.53; N, 20.28. Found

C, 36.71; H, 3.90; N, 19.19). Although NBD would be a useful tool to obtain details of cellular

127 internalization and trafficking of the PPC, further attempts to conjugate NBD were not made due to difficulties of the syntheses.

Figure 6.16. 1H NMR spectra of 6-NBD ligand (Top) and Monoplatin-NBD (Bottom). Due to difference

1 in solubility, H NMR spectrum of 6-NBD was obtained in CD3OH while the spectrum of Monoplatin-NBD was obtained in D2O.

128

6.5 Synthesis of Fluorine-18 Labelled Cisplatin

Positron Emission Tomography (PET) is a non-invasive quantitative medical imaging technique that takes advantage of positron decay to offer in vivo imaging of labelled compound or biomolecules (218,219). This technique allows one to obtain quantitative 2D and 3D biochemical and physiological information through the use of positive emitting radioelements such as 11C, 13N,

15O, and 18F (220). Development of a new imaging probe is rapidly expanding since it provide a wealth of information for drug development and mechanism investigation including data on target engagement, as well as pharmacokinetic and pharmacodynamic properties of the drug (221).

The most predominate isotope for radiolabeling of biomolecules for PET imaging is

Fluorine-18 (18F) because of its positron emitting property and favorable half-life of 109.8 min.

This half-life is significantly longer than 11C, 13N, and 15O, which allows enough time prepare the labelled complex with even those involving multiple steps. The conversion of an alcohol to a sulfonate ester (mesylate, tosylate, triflate) and reacting with 18F ion is a commonly used method for fluorine-18 labelling (222).

Cisplatin is a chemotherapeutic agent commonly used in the treatment of a wide variety of malignant tumors. DNA is known to be an important cellular target of cisplatin in living cells but much of the cellular and molecular biology of cisplatin action still remains unknown. Many attempts were made to obtain a cellular imaging of cisplatin by labelling the compounds with fluorophores; however, they do not provide in vivo imaging capacity and they may not represent cisplatin accurately due to relatively large structures of the fluorophores. To study the trafficking and localization of cisplatin distribution in living animals by in vivo PET imaging, an attempt were made to synthesize 18F labelled cisplatin. 18F labelling and PET imaging will be done with our collaborator, Dr. Zweit (VCU molecular imaging center) with expertise in molecular imaging.

129

Figure 6.17. Proposed synthetic scheme of fluorine-18 labelled cisplatin

Intermediate cis-Pt[Cl2(butanolamine)(NH3)] was obtained by using synthetic approach described in Figure 6.17; however, tosyl- or triflic- compounds were unable to be isolated due to high reactivity of the compounds. Further attempts are suggested in a glove box, where inert atmosphere is provided.

Experimental

Cisplatin was synthesized by using a method published previously (108).

Synthesis of K[PtCl3(NH3)] (TCAP)

Cisplatin (1g) was suspended in water (20 ml) and concentrated HCl (10 ml) was added. The mixture was heated under reflux for 8 hours. The clear, orange colored solution was allowed to cool to room temperature overnight. The mixture was filtered to remove any remaining cisplatin.

The filtrate was then reduced to approximately 5 mL under vacuum and stored in the refrigerator overnight. Any precipitant (cisplatin) was filtered off. The ammonium salts (NH4[PtCl3(NH3)] and

130

NH4[PtCl4]) were converted to potassium salts using a strong acidic cation exchanger. The exchanger was converted into K+ form by rinsing with a 2 molar KCl solution. The column was subsequently washed with water and the combined eluents were evaporated to dryness. If further purification was necessary, the product was recrystallized in water/methanol (4:1).

Synthesis of cis-Pt[(Cl)(I)(butanolamine)(NH3)

The synthesis of this compound was achieved by a modification of a published procedure (223).

K[PtCl3(NH3)] (0.84 mmol) was dissolved in 2 ml of a 15 mM aqueous solution of KCl. KI (1.92 mmol) was added to the K[PtCl3(NH3)] solution, followed by the butanolamine (0.9 mmol). A yellow precipitate was formed subsequently upon adding and the reaction was allowed to react for

30 minutes to allow the reaction to complete. Formed precipitant was collected, washed with cold water, cold methanol and ether, and then dried under vacuum overnight.

1 (Yield 35%). H NMR (CD3COCD3): δ 4.4(t,2H), 3.5(t, 2H), 1.8(q, 2H), 1.5(q, 2H)

Synthesis of cis-Pt[Cl2(butanolamine)(NH3)

Cis-[PtICl(butanolamine)(NH3)] (0.2 mmol) was suspended in 5 ml of H2O and 1.6 equivalents of silver nitrate was added to the suspension. It was then stirred overnight in the dark at room temperature. Formed AgCl was filtered through celite and 0.5 ml of HCl (˜12 M) was added to the filtrate. The solution was stirred at room temperature for 45 min and evaporated to dryness. The light yellow precipitate suspended with methanol and collected through filter.

1 (Yield: 83%). H NMR(D2O): δ 4.5(t,2H), 3.5(t, 2H), 1.8(q, 2H), 1.5(q, 2H) . Elemental Anal calc’d for C4H14Cl2N2OPt- C 12.91, H 3.79, N 7.53. Found: C13.15, H 3.59, N 7.31.

131

6.6 Platinum Recovery

Platinum is an expensive material and they are recovered from the laboratory wastes to reproduce

K2PtCl4, a substance used as a starting material in the synthesis of anticancer drugs.

Figure 6.18. Recovery of K2PtCl4 from platinum waste.

Preparation of Pt(0)

Excess NaOH was added to the liquid waste containing platinum until pH was higher than 10.

Then the solvent was evaporated under heat. The dried platinum was combined with solid waste containing platinum, then they were heated to redness with Meker burner until fuming stops. The ash was washed with warm water until the filtrate was clear. (Color of final product: black)

132

Preparation of Hexachloroplatinic(IV) Acid [H2PtCl6] Solution

Aqua regia was prepared freshly by mixing concentrated nitric acid and hydrochloric acid in a volume ratio of 1:3. Pure platinum metal was dissolved in gently heated aqua regia (30 mL per 1 g of platinum solid). The solution was cooled to the ambient temperature then filtered through filter paper to remove any impurity. To precipitate K2PtCl6, KCl was added (1g ash: 1g KCl) to the solution. A bright yellow precipitate of K2PtCl6 formed almost instantaneously and continued throughout the addition of KCl. The solvent was reduced to 1/3 of the original volume by heating then it was cooled in fridge to allow more precipitation. The precipitants were collected by filter then washed with acetone and ether.

Preparation of Potassium Tetrachloroplatinate(II) [K2PtCl4]

K2PtCl6 was suspended in 0.1 M HCl (40g / 400mL) and heated to 65 °C. 0.5 equivalents of hydrazine dihydrochloride dissolved in water was added dropwise to K2PtCl6 suspension over 2 hours. Then the mixture was allowed to react for additional 2 hours at 65° C. After 4 hours, only

a very small portion of solid K2PtCl6 remained in a very red solution. The platinum solution was filtered while hot to remove the unreacted K2PtCl6. Then the filtrate was concentrated by heating and any yellow precipitants were filtered out. This procedure was repeated until no yellow precipitant was observed. The volume of the solution was reduced close to dryness then then cooled in the fridge to force precipitation of K2PtCl4. The resultant red crystalline K2PtCl4 was filtered then washed with acetone and allowed to air dry in a 50 °C oven.

Total Recovery: 90 g of K2PtCl4

133

6.7 Contributions

All of the work in this chapter was done by Daniel E. Lee except for platinum recovery, where

Samantha Tsotsoros contributed equally.

134

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