AGENTS FOR THE TREATMENT OF NON-SMALL CELL LUNG …

STRUCTURE–ACTIVITY RELATIONSHIP STUDIES OF HYBRID ANTITUMOR

AGENTS FOR THE TREATMENT OF NON-SMALL CELL LUNG CANCER

BY ZHIDONG MA

A Dissertation Submitted to the Graduate Faculty of

WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES

in Partial Fulfillment of the Requirements

for the Degree of

DOCTOR OF PHILOSOPHY

Chemistry

December 2009

Winston–Salem, North Carolina

Approved By:

Ulrich Bierbach, Ph.D., Advisor _____________________________ Examining Committee: James P. Vaughn, Ph.D. (Chair) _____________________________ Rebecca Alexander, Ph.D. _____________________________ Christa L. Colyer, Ph.D. _____________________________ S. Bruce King, Ph.D. _____________________________

I

ACKNOWLEDGEMENTS

The pursuit of a Ph.D. degree is a challenging journey, filled with success, failure,

friendship, loneliness, happiness, and frustration. It took me five years of my Ph.D. to

understand the significance of this degree. Reading journals, discussing with colleagues

and thinking independently in the field of chemistry and biomedical research helped me

understand medicinal chemistry and its great contributions to health care and society.

Therefore, I really want to thank my dissertation advisor, Professor Ulrich Bierbach, not

only for teaching me the detailed technical skills everyday in the past five years, but also

leading me to explore a new world of medicinal chemistry. Every time I had a question,

he is always there to help me out. I really appreciate his “tough love” as my advisor

helping me build my scientific career and his “soft love” as my friend helping me through

my Ph.D. years.

I would also like to acknowledge my committee members, Professor James

Vaughn, Professor Rebecca Alexander, Professor Christa Colyer, and Professor Bruce

King, for reading my dissertation and giving me constructive suggestions during my Ph.D.

study. I would also like to thank Dr. Cynthia Day for her great help with crystallography,

and Dr. Marcus Wright for his patient in teaching me NMR spectroscopy.

Our collaborators helped me new techniques and allowed me to perform my

experiments in their labs. I want to show my appreciation to our collaborators Dr.

Bradley Jones, Carl Young and Dr. Keith Levine at Research Triangle Institute for

allowing me to perform the ICP-MS experiment, Dr. Gregory L. Kucera and Gilda Saluta

at Wake Forest University Baptist Medical School for teaching me how to perform

cytotoxicity assays, and Dr. Trevor Hambley, Dr Nicole Bryce, and Alice Klein at The

II

University of Sydney for the opportunity to perform confocal fluorescence microscopy

studies in their lab.

My past and present lab members are really precious to me. We have shared so

much memorable time together. Thank you Dr. Rajsekhar Guddneppanavar, Dr. Jayati

Roy Choudhury, Lauren Eiter, Lu Rao, Leigh Ann Graham, Amanda Pickard, Partha

Choudhury, Gary Wilson, Chandra Vemulapalli, Tony DeMartino, Bart Steen, Josh

Dworkin, Jennifer Nguyen. Without your company, it would have been painful to be

alone in the lab.

My friends also play a very important role during my time in Wake Forest. We

always had a lot of fun together. I would like to thank my friends and chemist fellows

Michael Budiman, Subhasis De, Jiyan, Maben, Liqiong, Xiuli, Lei, Ranjan, Saurva,

Salwa, Erika and Veronica.

The funding was also important to me, without which it would have been

impossible for me to continue my research. I would like to thank the Richter Scholar

Award, the National Cancer Institute (Grant RO1 CA101880) and Wake Forest

University for supporting my Ph.D. work.

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This dissertation is dedicated to my beloved grandma, father, mother and brother.

IV

TABLE OF CONTENTS

Page

List of Tables VIII

List of Figures X

List of Abbreviation XVII

Abstract XXI

Chapter 1 Introduction 1

1.1 Cancer: Lung Cancer 1

1.1.1 Cancer Statistics 1

1.1.2 Non-Small-Cell Lung Cancer 1

1.2 Chemotherapy of Lung Cancer 2

1.2.1 DNA-targeted Cytotoxic Agents 3

1.2.2 Targeted Therapies 17

1.3 Platinum Antitumor Agents 18

1.3.1 Bifunctional Platinum Crosslinkers 18

1.3.2 Monofunctional Platinum Complexes 24

1.3.3 Other Non-classical Platinum Agents 25

1.3.4 Platinum–Intercalator Conjugates 28

1.4 Background and Significance of the Doctoral Research Program 30

1.4.1 The Development of PT-ACRAMTU 30

1.4.2 Synthesis of PT-ACRAMTU 32

1.4.3 Biological Activity and DNA Binding Mechanism 33

1.4.4 Goals of the Doctoral Research Program 35

V

Chapter 2 Synthesis, Characterization, and Biological Evaluation of

Guanidine Derivatives of ACRAMTU

2.1 Background 39

2.2 Experimental Section 43

2.2.1 Materials and Chemicals 43

2.2.2 Syntheses and Characterizations of Guanidine Derivatives 43

2.2.3 Crystallization and Data Collection 48

2.2.4 Biological Characterization: pKa Determinations, DNA

Binding Affinities and Cell Proliferation Assay 48

2.3 Results and Discussion 50

2.3.1 Two Unusual Reactivity Features of the

9-Aminoacridine Chromophore 50

2.3.2 Biological Evaluation: pKa Determinations, DNA

Binding Constants, and Cytotoxicities 57

2.3.3 Attempted Conjugation of ACRAMGU (38) with

Monofunctional Platinum 60

2.4 Conclusions 61

Chapter 3 Synthesis, Characterization, and Biological Evaluation of

Amidine Derivatives of PT-ACRAMTU

3.1 Background 63


3.2.1 Materials and Chemicals 64

VI

3.2.2 Synthesis and Characterization 65

3.2.3 Cell Proliferation Assay 69

3.2.4 Crystallization and Data Collection 70

3.2.5 NMR Spectroscopy 70

3.2.6 Liquid Chromatographic Separation–Mass

Spectrometry Detection 71

3.2.7 Confocal Fluorescence Microscopy 72

3.2.8 H460 Xenograft Study 73

3.2.9 Inductively Coupled Plasma–Mass

Spectrometry Detection of Platinum 74


3.3.1 Lewis Acid Assisted Addition Reaction 75

3.3.2 Design Rationale 77

3.3.3 Biological Activity 79

3.3.4 Kinetics of Platinum Binding with

Biologically Relevant Nucleophiles 81

3.3.5 Analysis of Platinum–Amino Acid Products 92

3.3.6 Determination of the Subcellular Localization of

Complex 54 99

3.3.7 Antitumor Activity and Systemic Toxicity 106

3.3.8 Distribution of Platinum in Murine Tissue Samples 108

3.4 Conclusions and Clinical Implications 112

VII

Chapter 4 Changing the Connection Site of the Linker in

PT-ACRAMTU from the 9-Position to the 4-Position

4.1 Background 116


4.2.1 Materials and Methods 117

4.2.2 Synthesis and Characterization 118

4.2.3 Biological Characterization: pKa Determinations

and Cell Proliferation Assay 121


4.3.1 Synthesis of Acridine-4-Carboxamides 123

4.3.2 Acid–Base Chemistry 124

4.3.3 Cytotoxicities 125

4.4 Conclusions 126

Chapter 5 Summary and Outlook 129

References 132

Appendix A 155

Appendix B 172

Appendix C 189

Scholastic Vita 191

VIII

LIST OF TABLES

Page

Table 1. Cytotoxicity of PT-ACRAMTU (31) in NSCLC. 33

Table 2. Summary of Chemical and Biological Characterization for

38, 40 and 43. 58

Table 3. Cytotoxicity Data (IC50, μM) for Platinum–Acridine Conjugates. 80

Table 4. Platinum Concentrations in Various Biological Tissues

(Tumors, Kidneys and Lungs) at Two Different Doses

(0.1 mg/kg and 0.5 mg/kg) Determined by ICP–MS

after 16 Hours of Tissue Digestion. 111

Table 5. Retention of Platinum Per Single Organ Calculated as

a Percentage of the Total Administered Doses (0.1 mg/kg

and 0.5 mg/kg) Determined by ICP–MS after 16 Hours of

Tissue Digestion. 112

Table 6. Acid–Base Properties and In Vitro Cytotoxicity (IC50, μM)

of Acridines and Platinum Acridines in HL-60 and NCI-H460

Cell Lines. 125

Table B1. Summary of X-ray Crystallographic Data for 1-[2-(Acridine-9-

ylamino)- ethyl]-1,3-dimethylguanidine Dihydrochloride (ACRAMGU·2HCl), 38. 172

Table B2. Bond Lengths [Å] and Angles [°] for 1-[2-(Acridine-9-

ylamino)-ethyl]-1,3-dimethylguanidine Dihydrochloride (ACRAMGU·2HCl), 38. 173-175

IX

Table B3. Summary of X-ray Crystallographic Data for (Z)-N-

[1-(Acridin-9-yl)-3-methylimidazolidin-2-ylidene]methanaminium Hydrochloride, 40. 176

Table B4. Bond Lengths [Å] and Angles [°] for (Z)-N-

[1-(Acridin-9-yl)-3-methylimidazolidin-2-ylidene]methanaminium Hydrochloride, 40. 177

Table B5. Summary of X-ray Crystallographic Data for 2′-

(1H-Imidazol-1-yl)-8′-methyl-7′,8′-dihydro-6′H,10H-spiro- [acridine-9,4′-imidazo[1,2-a][1,3,5]triazine], 42. 178

Table B6. Bond Lengths [Å] and Angles [°] for 2′-(1H-Imidazol-

1-yl)-8′-methyl-7′,8′-dihydro-6′H,10H-spiro-[acridine- 9,4′-imidazo[1,2-a][1,3,5]triazine], 42. 179-180

Table B7. Summary of X-ray Crystallographic Data for 1-(Acridin-

9-yl)-3-methylimidazolidin-2-imine Hydrobromide, 43. 181 Table B8. Bond Lengths [Å] and Angles [°] for 1-(Acridin-

9-yl)-3-methyl-imidazolidin-2-imine Hydrobromide, 43. 182 Table B9. Summary of X-ray Crystallographic Data for

[Pt(EtCN)(C19H22N4)Cl2], 50. 183 Table B10. Bond Lengths [Å] and Angles [°] for

[Pt(EtCN)(C19H22N4)Cl2], 50. 184-185

Table B11. Summary of X-ray Crystallographic Data for

[PtCl(en)(C19H23N4)](NO3)2, 54. 186 Table B12. Bond Lengths [Å] and Angles [°] for

X

[PtCl(en)(C19H23N4)](NO3)2, 54. 187-188 Table C1. Platinum Concentrations in Various Biological Tissues

(Tumors, Kidneys and Lungs) at Two Different Doses (0.1 mg/kg and 0.5 mg/kg) Determined by ICP–MS After 16 Hours of Digestion. 189

Table C2. Masses of Various Biological Tissues (Tumors,

Kidneys and Lungs). 190

LIST OF FIGURES

Page

Figure 1. Timeline: The history of chemotherapy. 3

Figure 2. Watson–Crick base pairs (GC and AT) with the major

and minor grooves highlighted. 4

Figure 3. Classification of DNA–interactive agents and their

molecular interaction with DNA. 6

Figure 4. Ethidium, doxorubicin and echinomycin are examples of a

simple intercalator, complex intercalator and polyfunctional

intercalator, respectively. 7

Figure 5. Natural products found as DNA binders. 9

Figure 6. Minor groove hydrogen bonding patterns of Watson–Crick

base pairs. 10

Figure 7. The pyrimidine triple helix motif. 11

Figure 8. Chemical structure of a PNA. 12

Figure 9. The development of N-mustard-based cytotoxic agents. 13

XI

Figure 10. Monoalkylation and cross-linking chemistry of alkylating agents. 14

Figure 11. Structure of bleomycin. 15

Figure 12. DNA degradation products after bleomycin-induced

strand cleavage. 17

Figure 13. Mechanism of action of cisplatin. 19

Figure 14. Binding modes between cisplatin and DNA

(intrastrand and interstrand cross-links) 20

Figure 15. The current platinum drug “family tree” and generalized

rationale underlying their development. 22

Figure 16. Examples of bioactive monofunctional platinum complexes. 25

Figure 17. Interstrand cross-links for cisplatin (left)

and transplatin (right). 26

Figure 18. Structure of the trinuclear platinum complex BBR3464. 27 Figure 19. Schematic drawing of a platinum–intercalator conjugate. 28 Figure 20. Structures of platinum–intercalator conjugates. 29 Figure 21. Design of PT-ACRAMTU. 31 Figure 22. Synthesis of PT-ACRAMTU. 32 Figure 23. Potential reaction pathways of Pt–guanine adduct formation. 34

Figure 24. Structure of ACRAMTU and its guanidine analogue. 39

Figure 25. Slow decomposition of PT-ACRAMTU due to nucleophilicity

of coordinated thiourea sulfur. 41

Figure 26. Mechanistic implications of the kinetic cis-effect. 42

Figure 27. First attempt to generate ACRAMGU (38) by desulfurating 37

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with phosgene which leads to unexpected cyclized compound 40. 51

Figure 28. Second attempt to generate ACRAMGU (38) by sequential

replacement of imidazole in 41 by 36 and methylamine, which

leads to unexpected spiro compound 42. 52

Figure 29. Third attempt to generate ACRAMGU (38) using cyanogen

bromide, followed by addition of methylamine, which leads to cyclized compound 43. 53

Figure 30. Synthetic scheme for ACRAMGU, 38. 54

Figure 31. Proposed mechanism of formation of 42. 55

Figure 32. Crystal structures of 40 (chloride salt, left) and

43 (bromide salt, right). 56

Figure 33. Crystal structures of 42 (left) and protonated 38

(right, anions not shown).. 57

Figure 34. pH titrations for compounds 38 and 40 monitored

by UV-visible spectroscopy (pH range 2-12). 58

Figure 35. Reactivity of ACRAMGU (38) with activated [Pt(en)Cl2]. 60

Figure 36. Alternative strategies for generating platinum–imino

analogues of PT-ACRAMTU (31). 63

Figure 37. Test reaction for addition of secondary amine to

platinum-bound nitrile. 76

Figure 38. Molecular structure of trans-[PtCl2(EtCN)(N-

[2-acridin-9-ylamino]ethyl]-N-methylpropionamidine)]

50 with selected atoms labeled. 77

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Figure 39. Synthesis of target compounds. 78

Figure 40. Molecular structure of 54 with selected atoms labeled. 79

Figure 41. Kinetic study using time-dependent 1H NMR spectra for

the reaction of PT-ACRAMTU (31) and complex 54 with 5´-GMP monitored for aromatic guanine and acridine protons giving signal assignments. (A) Reaction scheme with important atoms labeled. (B) Stacked plot for the reaction monitored for PT-ACRAMTU (31). (C) Stacked plot for the reaction monitored for 54. 81-82

Figure 42. Progress of the reaction of PT-ACRAMTU (31) (circles) and

54 (triangles) with the mononucleotide 5´-GMP at 37 oC

monitored by 1H NMR spectroscopy. 83

Figure 43. Hydrolysis of complex 54´ detected by 2-D [1H-15N]

HMQC NMR spectroscopy giving cross-peak assignments. 85

Figure 44. Reaction of complex 54´ with 5´-GMP monitored by

2-D [1H-15N] HMQC NMR spectroscopy giving

cross-peak assignments. 86

Figure 45. Time-dependent 1H NMR spectra for the reaction of

PT-ACRAMTU (31) and complex 54 with N-AcCys.

(A) Reaction scheme with protons monitored highlighted. (B) Stacked plot for the reaction monitored for PT-ACRAMTU (31). (C) Stacked plot for the reaction monitored for 54. 88-89

XIV

Figure 46. Progress of the reactions of 54 (blue circle) and

PT-ACRAMTU (red square) with one equivalent of N-AcCys at 25 oC monitored by 1H NMR spectroscopy. 90

Figure 47. (A) Reverse-phase HPLC elution profile monitored at 413 nm

of a mixture of complex 54 incubated with N-AcCys. Positive-ion electrospray mass spectra of adducts P2 (B) and P3 (C) showing peaks for the molecular ion, [P3]+ (m/z 725.2) and [P2-2H]2+ (m/z 643.7, z = 2) and fragments resulting from in-source collisionally activated dissociation (CAD). 93-94

Figure 48. (A) Reverse-phase HPLC elution profile monitored at 413 nm

of a mixture of PT-ACRAMTU 31 incubated with N-AcCys. Positive-ion electrospray mass spectra of adducts P1´ (B) and P2´ (C) showing peaks for the molecular ion, [P1´-2H]2+ (m/z 660.2, z = 2) and [P2´]+ (m/z 903.4) and fragments resulting from in-source collisionally activated dissociation (CAD). 96-97

Figure 49. Pathways for reaction of PT-ACRAMTU (31) and 54

with N-AcCys. 98 Figure 50. Fluorescence microscopy image of A2780 cells treated with

LysoTracker Red DND-99 (left) and MitoTracker Green FM (right). 100

Figure 51. Representative fluorescence microscopy image (left) of

A2780 cells incubated with complex 54 for 10 min. Enlarged image (right) of a single cell . 101

XV

Figure 52. Confocal fluorescent microscopy images of A2780 cells

treated with complex 54 while co-stained with MitoTracker Green FM (top) and LysoTracker Red (bottom), respectively. and (A) show images taken through blue emission filter, (II) shows an image taken through green emission filter, (III) shows superpositions of blue and green images, (B) shows an image taken through red emission filter, and (C) shows superpositions of blue and red images. 103

Figure 53. Representative fluorescence microscopy images of A2780

cells incubated with complex 54 alone (left) and co-treated with hydrogen peroxide (right) for 10 mins. 105

Figure 54. Evaluation of 55 against H460 NSCLC tumors xenografted

into nude mice. 107 Figure 55. Platinum concentrations in various biological tissues (tumors,

kidneys and lungs) at two different doses (0.1 mg/kg and 0.5 mg/kg) determined by ICP–MS after 16 hours of tissue digestion. 109

Figure 56. Retention of platinum in each organ calculated as percentage of

the total administered doses (0.1 mg/kg and 0.5 mg/kg) determined by ICP–MS after 16 hours of tissue digestion. 111

Figure 57. Changing 9-amino acridine into acridine-4-carboxamide. 117 Figure 58. Synthesis of acridine-4-carboxamides 57 and 60 and platinum

conjugate 58. 124

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Figure A1. 1H NMR (top) and 13C-{1H} NMR (bottom) spectra of

compound 38 in D2O. 155 Figure A2. 1H NMR (top) and 13C-{1H} NMR (bottom) spectra of


compound 42 in DMF-d7. 157 Figure A4. 1H NMR (top) and 13C-{1H} NMR (bottom) spectra of


compound 45 in CDCl3. 159

Figure A6. 1H NMR (top) and 13C-{1H} NMR (bottom) spectra of

compound 46 in CDCl3. 160 Figure A7. 1H NMR (top) and 13C-{1H} NMR (bottom) spectra of

compound 47 in MeOH-d4. 161 Figure A8. 1H NMR (top) and 13C-{1H} NMR (bottom) spectra of

compound 52 in D2O. 162 Figure A9. 1H NMR spectrum of compound 52′ in MeOH-d4. 163 Figure A10. 1H NMR (top) and 13C-{1H} NMR (bottom) spectra of


compound 54 in DMF-d7. 165 Figure A12. 1H NMR spectrum of compound 54′ in DMF-d7. 166 Figure A13. 1H NMR (top) and 13C-{1H} NMR (bottom) spectra of

XVII

compound 55 in DMF-d7. 167 Figure A14. 1H NMR (top) and 13C-{1H} NMR (bottom) spectra of

compound 57 in DMSO-d6. 168 Figure A15. 1H NMR (top) and 13C-{1H} NMR (bottom) spectra of

compound 58 in DMF-d7. 169 Figure A16. 1H NMR spectrum of compound 59 in MeOH-d4. 170 Figure A17. 1H NMR (top) and 13C-{1H} NMR (bottom) spectra of

compound 60 in D2O. 171

LIST OF ABBREVIATIONS

A Aden(os)ine ACRAMTU 1-[2-(Acridin-9-ylamino)ethyl]-1,3-

Dimethylthiourea

ACRAMGU 1-[2-(Acridine-9-ylamino)ethyl]-1,3-

dimethylguanidine

AMD473 cis-Amminedichloro(2-methylpyridine)platinum(II)

B Nucleobase

BBR3464 [(trans-PtCl(NH3)3(trans-Pt(NH3)2-

(NH2(CH2)6NH2)2))](NO3)4

BOC tert-Butylcarbamate

C Cytosine

Carboplatin cis-Diammine-1,1-

cyclobutanedicarboxylatoplatinum(II)

CAD Collisionally activated dissociation

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CML Chronic myeloid leukemia

Cisplatin cis-Diamminedichloroplatiunm(II)

COSY Correlated spectroscopy

DACA N-[2-(Dimethylamino)ethyl]acridine-4-carboxamide

DACH 1,2-Diaminocyclohexane

DSS 3-(Trimethylsilyl)-1-propanesulfonic acid sodium

salt

dGuo 2′-Deoxyguanosine

DNA Deoxyribonucleic acid

dNTP Deoxyribonuleotide triphosphate

ds Double-stranded

DMF N,N′-Dimethylformamide

DMS Dimethylsulfide

DMSO Dimethylsulfoxide

en Ethane-1,2-diamine, ethylenediamine

enplatin Ethylenediaminedichloroplatinum(II), [PtCl2(en)]

EGFR Epidermal growth factor receptor

EtCN Propionitrile

FDA Food and Drug Administration

G Guan(os)ine

GMP Guanosine monophosphate

GSH Glutathione

HIV Human immuno deficiency virus

XIX

HOMO–LUMO Highest occupied molecular orbital–lowest

unoccupied molecular orbital

HPLC High-performance liquid chromatography

HSQC Heteronuclear single quantum coherence

Hp Hydroxypyrrole

ip Intraperitoneally

IC50 Inhibitory concentration

ICP Inductively coupled plasma

ICP–MS Inductively-coupled plasma electrospray mass

spectrometry

Im Imidazole

JM216 Bis-acetatoamminedichlorocyclohexylamine

platinum(IV)

JM473 cis-Amminedichloro-2-methylpyridineplatinum(II)

Kapp Apparent binding constant

KOH Potassium hydroxide

LC-ESMS Liquid chromatography-electrospray mass

Spectrometry

MTD Maximum tolerated dose

N-AcCys N-acetylcysteine

NER Nucleotide excision repair

NMR Nuclear magnetic resonance

NSCLC Non-small-cell lung cancer

XX

NOESY Nuclear Overhauser enhancement spectroscopy

Oxaliplatin trans-L-Diaminocyclohexaneoxalatoplatinum(II)

ppb Parts per billion

ppm Parts per million

PBS Phosphate-buffered saline

Picoplatin cis-Amminedichloro-2-methylpyridineplatinum(II)

PNA Peptide nucleic acid

PT-ACRAMTU [Pt(en)Cl(ACRAMTU)](NO3)2

Py Pyridine

ROS Reactive oxygen species

Satraplatin bis-Acetatoamminedichlorocyclohexylamine

platinum(IV)

SAR Structure−activity relationships

SCLC Small-cell lung cancer

SEM Standard error of the mean

SOPs Standard Operating Procedures

T Thymi(di)ne

TFOs Triplex-forming oligonucleotide

THF Tetrahydrofuran

TMS Trimethylsilane

Transplatin trans-Diamminedichloroplatinum(II)

WATERGATE Water suppression by gradient-tailored excitation

WHO World Health Organization

XXI

ABSTRACT

Structure–Activity Relationship Studies of Hybrid Antitumor Agents for the Treatment of

Non-Small-Cell Lung Cancer

By

Zhidong Ma

Dissertation under the direction of Ulrich Bierbach, Ph. D.

Associate Professor of Chemistry

DNA-directed chemotherapies continue to play an important role in modern

oncology. The cytotoxic complex, [PtCl(en)(ACRAMTU)](NO3)2 (en = ethane-1,2-

diamine; ACRAMTU = 1-[2-(acridine-9-ylamino)ethyl]-1,3-dimethylthiourea) (PT-

ACRAMTU, 31), is a dual platinating/intercalating DNA binder that, unlike clinical

platinum agents, does not induce DNA cross-links. The research in this dissertation was

concerned with establishing structure–activity relationships (SAR) in this novel class of

DNA-targeted antitumor agents with the ultimate goal of improving the biological

activity of the prototypical agent, PT-ACRAMTU.

Two ways were explored of tuning the reactivity and target interactions of

platinum, which was considered critical in changing the pharmacological properties of

this pharmacophore. The first approach involved a structurally minimally invasive

modification of the prototype to enhance its chemical stability and reactivity with DNA.

This was achieved by introducing a new inert nonleaving group (imino group) in place of

thiourea. The first attempt involved the synthesis of a guanidine analogue of ACRAMTU,

1-[2-(acridine-9-ylamino)ethyl]-1,3-dimethylguanidine (38), by adding N-acridin-9-yl-N'-

XXII

methylethane-1,2-diamine (36) to a Boc-activated carbodiimide (Me-N=C=N-Boc),

obtained by desulfuration of N-methylthiourea (44) with HgCl2. While the study was able

to delineate unusual pathways to novel cyclic and spirocyclic acridine derivatives, the

ultimate goal of generating a guanidine analogue of PT-ACRAMTU was not reached due

to the chelating properties of the guanidinato group (verified by 195Pt NMR). Instead,

platinum-mediated amidination chemistry afforded two amidine derivatives of PT-

ACRAMTU (54 and 55) with greatly enhanced activity (IC50 values of 26 nM and 28 nM,

respectively) in H460 non-small-cell lung cancer (NSCLC). The amidination reaction

involved addition of the secondary amine in 36 across the activated CN bond of

platinum-bound propionitrile (EtCN). Complex 54 proved to be a more efficient DNA

binder (t1/2 = 65 min) than PT-ACRAMTU (31) (t1/2 = 234 min), and showed

considerably reduced reactivity with N-acetylcysteine compared to PT-ACRAMTU,

based on a mechanistic study using time-dependent 1H NMR and 2-D HMQC NMR

spectroscopy, as well as in-line liquid chromatography–electro-spray mass spectrometry

(LC–ESMS). A cellular imaging study using confocal fluorescence microscopy was

performed to study the subcellular distribution of complex 54. The results show higher

cellular levels of 54 than of PT-ACRAMTU and suggest that the drug reaches the nucleus,

although a significant amount of compound seems to be trapped in the lysosomes. A

H460 mouse xenograft study showed complex 55 slows tumor growth by 40% when

administered at a dose of 0.5 mg/kg, but the compound was quite toxic and caused weight

loss in the animals. The concentrations of platinum detected using inductively-coupled

plasma electrospray mass spectrometry (ICP–MS) in various biological tissues recovered

from the euthanized mice reveal that, while compound 55 reaches the tumors, most of the

XXIII

detected platinum accumulated in the kidneys, the major organ of excretion. The high

level of platinum in the kidneys most likely contributes to the toxicity observed in the

treated animals. Complex 55 is the first non-cross-linking platinum agent endowed with

promising activity in NSCLC.

In the second approach, novel thiourea- and guanidine-modified acridine-4-

carboxamides and a corresponding platinum–intercalator conjugate have been

synthesized and evaluated as cytotoxic agents. The point of attachment of the platinum-

modified linker was changed from the 9-position to the 4-position of acridine to alter the

DNA binding of the conjugate and its cytotoxicity. The IC50 value of 2.4 μM determined

in H460 lung cancer cells for [PtCl(en)(N-(2-(1,3-dimethylthioureido)ethyl)acridine-4-

carboxamide)](NO3)2 (58) indicates that this strategy has no advantage over the 9-

aminoacridine-based prototype.

1

CHAPTER 1

INTRODUCTION

1.1 Cancer: Lung Cancer

1.1.1 Cancer Statistics

Lung cancer has become the number one cause of death from cancer each year,

accounting for one third of all cancer deaths worldwide. Although its incidence has

decreased significantly in men by 37% between 1990 and 2005, because of the reduction

in tobacco use during the past 50 years, lung cancer remains the leading cause of cancer-

related mortality in the United States. Statistically, a total of 219,440 new lung cancer

cases and 159,390 deaths from this disease are projected to occur in the United States in

2009 [1, 2].

The two main types of lung cancer are small-cell lung cancer (SCLC) and non-

small-cell lung cancer (NSCLC), which can be differentiated by the size and appearance

of the malignant cells observed histologically under a microscope [1]. They can also be

distinguished primarily based on their clinical behaviors: SCLC has a particular

aggressive clinical course with widespread early metastasis and, in contrast to NSCLC,

will show a better response to cytotoxic chemotherapy and radiation [3, 4].

1.1.2 Non-Small Cell Lung Cancer

While SCLC accounts for approximately 15% of new cases of lung cancer

diagnosed annually and for up to 25% of lung cancer deaths each year [3], NSCLC,

which includes three main sub-types (adenocarcinomas, squamous carcinomas, and large-

cell carcinomas), comprises 80% of lung cancers [5]. Most patients with advanced

2

metastatic NSCLC, in which cancer cells have gained the ability to invade normal

tissue(s) away from their tissue of origin, have a median survival after diagnosis of 4–5

months and a year survival of less than 10%, if left untreated [6].

1.2 Chemotherapy of Lung Cancer

Prior to the 1950s, surgery was the only option in lung cancer treatment.

Radiation therapy became a valuable tool to control local and regional disease after 1960

[7]. However, it became clear that more effective treatments for most patients needed to

reach every affected organ in the body. Although the cause of cancer is not yet well

understood, it is obvious that various mutations in the nuclear environment occur during

the formation of cancer, and some anticancer drugs aim to target a cancer cell’s specific

pathways to minimize the adverse effects on normal cells [8]. These mutations, including

translocations, deletions, amplifications, as well as other changes, may have a severe

impact on normal nuclear processes, such as DNA replication, repair, and expression [8].

Therefore, small-molecule drugs, biological molecules, and immune-mediated therapies

have become the focus of current efforts to treat cancer with minimal side effects to

normal tissues. The era of chemotherapy began in the 1940s with the use of nitrogen

mustards and antifolate drugs [9]. Cancer drug development since then has transformed

from a low-budget, government-supported research to a multi-billion dollar industry [7].

Drug design, which includes the traditional cytotoxic agents as well as the targeted

therapies, with the assistance of established structure–activity relationship (SAR) and

high-throughput industrial screening, remains a challenging field for scientists worldwide

(Figure 1) [7].

3

Figure 1. Timeline: The history of chemotherapy. Taken with permission from reference [7].

1.2.1 DNA-targeted Cytotoxic Agents

Double-stranded B-form DNA consists of four building blocks called nucleotides,

each of which contains a nucleobase, a five-membered deoxyribose sugar ring, and a

phosphate group. The right-handed double helix has approximately 10 base pairs per turn

with the two spiral strands intertwined in an anti-parallel fashion and connected to one

other through hydrogen bonds between adenine (A) and thymine (T) and between

guanine (G) and cytosine (C) (Watson–Crick base pairing, Figure 2). In a Watson–Crick

base pair, the two deoxyribose residues point in the same direction, which causes gaps in

between the sugars of consecutive base pairs and creates continuous indentations in DNA.

The continual indentations wind along, parallel to the sugar–phosphate backbone, to

generate two grooves termed the major and the minor groove.

4

Figure 2. Watson–Crick base pairs (GC and AT) with the major and minor grooves highlighted.

The DNA grooves have multiple receptor sites harboring nucleophiles for DNA-

interactive drugs (Figure 2) [10], of which the purine nitrogens are particularly

susceptible to modification. In the major groove, guanine-N7 is the most reactive site,

while the methyl group of thymine, O6 of guanine, and N6 of adenine are also recognized

by DNA-binding proteins and small molecules [11, 12]. Nucleophilic attack in the minor

groove mainly occurs at the purine-N3 [10, 13].

There is a recent study by the World Health Organization (WHO) showing that of

the 17 high-priority clinical anticancer drugs, 7 are DNA-binding agents [14]. Agents that

associate with DNA covalently or non-covalently are considered potential inhibitors of

mitotic progression and inducers of apoptotic cell death, and, thus, are of interest in

cancer chemotherapy [15]. DNA as carrier of genetic information is a major target for

drug interaction due to its unique function in transcription (gene expression) and DNA

replication, a major step in cell growth and division. Therefore, after several decades of

successful clinical applications of cytotoxic agents, the targeting of DNA as well as

specific DNA-associated enzymes continues to be an important approach in designing

N1

N3

N7O

N

N9O

HO

HON1

N3

N9

N7N

O

HO

HO

O

OH

OHN1

N3

O

OO

OH

OHN1

N3

N

O

HH

AT

Minor groove

Major groove

H

HH

HH

GC

Minor groove

Major groove

H

H1'H1'

H1'H1'

5

cancer therapies [8, 9]. The ultimate goal is to disrupt vital cellular processes at the

nuclear level to trigger downstream events ultimately leading to cancer cell death.

Numerous cancer therapeutics produce cytotoxic responses by damaging nuclear DNA,

thereby interfering with the replication and transcription of the nucleic DNA.

However, one has to be aware that DNA-targeted cytotoxic agents exert their

effects on all proliferating cells, both normal and cancerous. In other words, cytotoxic

agents exhibit a selective antiproliferative action rather than selective anticancer

properties [16]. It is then understandable that in the development of cytotoxic anticancer

agents, toxic side effects and drug resistance are almost inevitable. On the other hand,

cytotoxic agents are still entering the clinic and are gaining regulatory approval because

of their significant effectiveness, which is the result of cytotoxic damage to rapidly

replicating cells, such as abnormal cancer cells.

The interactions between small molecules and DNA have been broadly classified

into two major groups: noncovalent and covalent interactions. Intercalators (insertion of

planar, or approximately planar, aromatic ring systems into base-pair steps) and code-

reading agents (groove binding with the edges of the base pairs in the major or minor

groove) belong to the first group, while alkylating agents, DNA–DNA cross-linkers,

DNA-cleaving agents, and metalating agents belong to the second group [17].

(Metalating agents will be discussed in detail in Section 1.3, Platinum Antitumor Agents)

Figure 3 shows various types of drug-induced DNA modifications including, from

left to right, cross-links, intercalation, DNA strand cleavage, and code-reading

interactions [17]. The concept of targeting DNA–enzyme complexes is also illustrated.

The following section gives an overview of various kinds of DNA targeting agents along

6

with their (potential) therapeutic applications, while some of the agents’ modes of action

remain to be elucidated [18].

Figure 3. Classification of DNA-interactive agents and their molecular interaction with DNA. Taken with permission from reference [17].

Group I: Noncovalent interactions

Group Ia. Intercalators

Intercalators are usually small organic molecules, characterized as planar

chromophores (usually fused aromatic rings) that insert between adjacent nucleobase

pairs. Polarization forces, van der Waals interactions, and interactions between the

HOMO–LUMO π-orbitals (highest occupied molecular orbital–lowest unoccupied

molecular orbital) of the planar chromophore and nucleic acid base pairs are the three

major factors contributing to the binding affinity of intercalators [19]. Intercalators

lengthen and unwind the DNA helix in order to π stack between two base pairs, thereby

distorting the DNA backbone conformation and interfering with DNA–protein

interactions.

DNA-DNA Crosslinker

Intercalator

Double-strandedbreak

Code reading Protein-DNAcomplex

7

Typical intercalators include: (i) simple intercalators with one planar

chromophore, (ii) complex intercalators with planar chromophore attached to other

groups (such as sugar residues or peptide units), and (iii) polyfunctional intercalators with

multiple planar chromophores.

Figure 4. Ethidium, doxorubicin and echinomycin are examples of a simple intercalator, complex intercalator and polyfunctional intercalator, respectively.

In Figure 4, ethidium bromide (1) is one of the classic simple DNA-intercalating

agents that has been known for many years for its ability to inhibit nucleic acid synthesis

in a variety of parasitic organisms [20]. Doxorubicin (2), on the other hand, represents a

complex intercalator, which has made a major impact in the treatment of cancer patients

since the 1960s [21]. It produces DNA strand breaks by inhibiting the reannealing

mechanism of topoisomerase II [22].

O

O

OH

OHMeO O

O OH

OH

O

OH H2N

NCH3

NH2H2N

N

NO

NH

O

O NMe

ONR

ONH

Me

OHN

ON

N

O

ONMe

O

S

NMe

OO

HN

Me

S

Doxorubicin, 2 Ethidium, 1

Echinomycin, 3

8

The idea of synthesizing bifunctional, or even polyfunctional, intercalators was

inspired by the possibility of enhancing the DNA binding compared to that of the

corresponding monomers. The quinoxaline antitumor antibiotics are a family of cyclic

depsipeptides that bisintercalate into DNA, positioning their peptide backbones in the

minor groove [23, 24]. They consist of two planar chromophores, tethered by flexible or

semirigid linker groups that are capable of simultaneously inserting into the DNA base

stack. Echinomycin (3) is a typical member of this family, which strongly binds DNA

and inhibits nucleic acid synthesis [25]. The syntheses of these bifunctional or even

polyfunctional intercalators have attracted great attention because of the notion that the

pharmacological activity of intercalating drugs can be greatly improved by significantly

increasing their DNA binding constants and slowing their DNA dissociation rates [26].

The mode of intercalation has now been established for a large number of

polycyclic aromatic systems [27]. Intercalation requires changes in the sugar–phosphate

torsional angles in order to accommodate the aromatic compound [26]. Although models

of duplex DNA interacting with intercalators at all possible sites suggest that this binding

mode is possible, experimental studies in solution indicate that even the simplest

intercalators appear to reach saturation at a maximum of one intercalator per two base

pairs. This observation has led to the neighbor exclusion principle: intercalators can bind

at alternate base-pair sites on DNA at most. In other words, when an intercalator binds at

one non-specific site, the exclusion principle states that binding another intercalator at a

directly adjacent site is prohibited [28].

Group Ib. Code-reading molecules

9

In 1964, Irving Goldberg and co-workers introduced the idea that small molecules,

such as actinomycin (4), may show sequence selectivity when interacting with DNA

(Figure 5) [29]. However, this notion was not appreciated until the first X-ray structure of

restriction enzyme bound to DNA stimulated an interest in small (synthetic) molecules

that are able to recognize DNA [30]. The discovery of minor-groove-recognizing

molecules, such as distamycin and netropsin (Figure 5) [30], paved the way for the design

and synthesis of DNA code-reading molecules that might ultimately target a unique

sequence, followed by the development of major-groove-recognition molecules [31, 32].

Figure 5. Natural products found as DNA binders.

The structural similarities shared by most of the minor-groove binders include a

positive charge, carbon–carbon single bond or amide-linked aromatic and/or

heteroaromatic rings (unlike the fused aromatic rings found in intercalators) and a

characteristic crescent shape.

Distamycin

NH

NH

HNO

HN

O

O

HN

O

HN

NH

NH2

NH

HN

O

O

HN

O

HN NH2

NH

NH

NH

H2N

Netropsin

O

N

O

NH2

O NHHN O

R

CHO

ON

O

N

O

NO

HNO

CH O

ON

O

N

O

NO

NHO

Actinomycin

Distamycin, 5

Actinomycin, 4 Netropsin, 6

10

Figure 6. Minor groove hydrogen bonding patterns of Watson–Crick base pairs.

The minor groove binders distamycin and netropsin (Figure 5) directly read out

hydrogen bonding as a specific form of DNA-sequence recognition. For example,

netropsin reads five A•T base-pair tracts, and binding to the minor groove is interrupted

by sequences having mixed A•T/G•C base pairs [30]. Considerable efforts have been put

into redesigning netropsin analogues so that they can read both A•T and G•C base pairs.

The new motif, side-by-side pyrrole–imidazole amino-acid pairing, is able to distinguish

all four Watson–Crick base pairs (A•T, T•A, G•C and C•G) [33]. In Figure 6 [33], the

NH of the carboxamides point towards the minor groove of the helix forming specific

hydrogen bonds with base pairs. The unsymmetrical pair, imidazole(Im)/pyridine(Py), is

used to distinguish GC from CG, while the newly designed pair,

hydroxypyridine(Hp)/pyridine(Py), distinguishes TA from AT. The design revealed that

H

HN

NO

NN

OH

O N

N

O N

N

O

NO

N

N N

NO

NHO

ON

N

ON

N

H

H

H

H

ON

H

H

H

H

H

ONHN

H5' 3'

Py/Im targets C

Py/Hp targets A

Hp/Py targets T

Im/Py targets G

G

T

A

C

11

the field of molecular recognition of DNA has matured from serendipity to successful

design [33].

It is well known that proteins can recognize and bind to DNA by reading

sequence code in either groove, and mostly through major groove. Non-peptidyl

compounds show the opposite preference, which means they bind through the minor

groove, allowing proteins to recognize the minor and major grooves simultaneously.

Accordingly, it would be interesting to design major-groove-binding molecules blocking

access to proteins that recognize the same groove, if sufficient affinity and sequence

selectivity can be obtained. Two types of major-groove binders have been developed, the

triplex-forming oligonucleotides (TFOs) [32] and the peptide nucleic acids (PNAs) [34].

Figure 7. The pyrimidine triple helix motif. Similar base triplets form TAT and C+GC. The additional pyrimidine is bound through Hoogsteen hydrogen bonds in the major groove to the complementary purine strand in the Waston–Crick base pair.

TFOs were first discovered in 1987 by Heinz Moser, who demonstrated that

oligodeoxyribonucleotides (15-mer) can form specific triple helical complexes in the

major groove of DNA under pH, temperature, and salt conditions not too different from

N

NN

N

N N N

O

O

NN O

O N N

N

O

NN N

ON

NN

N

O

N

H HHH

R

R

R

TAT base triplet

R

R

R

H

H H

H

H

H

H

H

C+GC base triplet

Major groove Major groove

12

those found in a living cell [35]. The sequence recognition in TFOs requires that T binds

AT and C binds GC (Hoogsteen TAT and C+GC triplexes) (Figure 7) [36]. It was

demonstrated that TFOs inhibit DNA binding proteins and modulate transcription in cell-

free systems [37, 38].

Peptide nucleic acids (PNAs) have been known for more than fifteen years and

were originally recognized as mimics of TFOs which are able to sequence-specifically

interact with double-stranded DNA [39]. They were built with uncharged, achiral, pseudo

peptide backbones and are therefore chemically more closely related to peptides and

proteins than to nucleic acids (Figure 8) [34]. Because of their excellent hybridization

properties, PNAs can also interact strongly with DNA strands to form stable duplex

hybrids [40]. Like the TFOs, PNAs in complex with double-stranded DNA effectively

block transcription initiation [41].

Figure 8. Chemical structure of a PNA. ‘B’ stands for nucleobase.

Even though both TFOs and PNAs recognize the major groove of DNA, there are

two major differences between them. First, the backbones of TFOs are modified

analogues of oliogonucleotides, whereas PNAs have peptide-like backbones. Second,

TFOs bind with the existing major groove of duplex DNA, whereas PNAs invade the

DNA helix to form a new hybrid duplex [42], as the result of the displacement of the non-

complementary DNA strand.

HN

N

OB

O

HN N

O

B

HN N

O

B

O

HN

ON-terminal

13

Group II: Covalent interaction

GroupIIa. Alkylating agents and DNA–DNA cross-linkers

Among the DNA interacting agents, covalent binders comprise the largest group.

The beginning of cancer chemotherapy is generally accepted to have been the

serendipitous discovery of the mustards family in the first half of the twentieth century

[9]. The nitrogen mustards were developed as derivatives of the sulfur-based mustard gas

(Figure 9) [9], which was first synthesized in 1886.

Figure 9. The development of N-mustard-based cytotoxic agents. Redrawn from reference [9] with modifications.

In 1946, it was discovered that the toxic effects of sulfur mustard were due to

DNA alkylation [43]. Figure 9 shows the historic development of the mustard family of

agents, from mustard gas to molecules that have shown progressively decreasing toxicity

S

Cl

Cl

N

Cl

Cl

N

Cl

Cl

HOOC

N

Cl

ClCOOHH2NN

Cl

Cl

PNH

O O

OH

O

OCl

Cl

Mustard gas(sulphur mustard)

Chlormethine(mustine)

Chlorambucil

Too toxic to be usedin humans

Aliphatic mustards havesufficient therapeuticindex to be used in humans

Aromatic mustards are less electrophilic and react with DNAmore slowly. Can be administeredorally

Melphalan

Represents an attempt to enhance cellular uptake through the phenylalanine-transport mechanism

Represents an attempt to release mustard agent through enzymatic degradation

An attempt to target estrogen-dependenttumor cells

Estramustine Cyclophosphamide

N

Mustard gas, 7 (sulfur mustard)

Chlormethine, 8 (mustine)

Chlorambucil, 9

Estramustine, 10 Cyclophosphamide, 11 Melphalan, 12

14

to normal cells, to a molecule design that allows mustard analogues to be targeted to

estrogen receptor positive tumor cells. It was realized that, if the electrophilicity of

mustards could be reduced, then less toxic drugs might be obtained that can be

administrated orally. This led to the development of the compounds chlormethine,

chlorambucil, melphalan, cyclophosphamide, and estramustine, which are still in clinical

use today. Melphalan is composed of a phenylalanine group attached to the mustard to

enhance the selective uptake by tumor cells. Estramustine is the combination of mustard

and an estrogen derivative [9].

Figure 10. Monoalkylation and cross-linking chemistry of alkylating agents.

Certain cancers, such as leukemias and lymphomas, with very high proliferative

rates compared with most normal tissues, are particularly susceptible to these agents. In

N-mustard-based alkylating agents, chloride is a good leaving group that favors

intramolecular nucleophilic attack of nitrogen to form a strained, highly reactive

aziridinium cation, which ring-opens in reactions with DNA nucleophiles (Figure 10)

[17]. The regiospecificity of alkylation of DNA by these nitrogen mustards is governed

primarily by the steric and electronic properties of DNA. Aziridinium cations readily

NCl

ClR

N

Cl

R DNA NDNA

ClR

MonoalkylatedDNA

NDNA

RDNA

NDNA

DNAR

CrosslinkedDNA

15

alkylate N7 of guanine to form a monoalkylation adduct because N7 on guanine is the

sterically least hindered site and has the most negative electrostatic potential, rendering

this nitrogen the strongest nucleophile in DNA [17]. This process can be repeated to form

the cross-linked DNA (Figure 10). Cross-links in DNA can occur either between two

complementary strands of DNA (interstrand, Figure 3), as generated by chlorambucil and

melphalan, or within one strand of DNA (intrastrand), as generated by cisplatin

(discussed in detail in Figure 15) [17].

b. DNA-cleavage agents

Bleomycin, unlike most DNA-damaging electrophiles, attacks neither the nucleic

bases nor the phosphate linkages, even though products of its attack include both nucleic

base derivatives as well as phosphomonoester termini [44, 45].

Figure 11. Structure of bleomycin.

N NO

HNO

CH3

O

HN

CH3

NHH2NCH3

O

N

S

N

S

O

NH

SH3C

CH3

HOHH

HOH

OH

N

NH

OHHO

OH

O

O

OH O

OH2N

OHOH

HN NH2

O

NH2H2N O

CH3 H

Bleomycin, 13

16

It is isolated as a copper complex from the culture medium of Streptomyces

verticillis, but administrated to patients with wide clinical antitumor use [46] in metal-

free form, which minimizes irritation at the site of injection (Figure 11).

The hypothesis that DNA damage is the major contribution of bleomycin’s

cytotoxicity is supported by the finding that cells treated with bleomycin contain cleaved

DNA with the same site specificity observed in vitro [47]. Involvement of iron and

reduced molecular oxygen in the mechanism of action of bleomycin indicated that

bleomycin might attack DNA through reactive oxygen species (ROS), such as hydroxyl

radical (•OH) [48]. The analysis of bleomycin reactions began with the discovery that in

the presence of oxidizing agents like hydrogen peroxide, bleomycin causes DNA

fragmentation [49]. Bleomycin was found to form a 1:1 complex with Fe(II) that reacts

with O2 [48]. Another study [50] showed that Fe(III)•bleomycin reacted with peroxide

yields a drug intermediate that cleaves DNA the same way Fe(II)•bleomycin + O2 does

[48].

The reactions between bleomycin and DNA are somewhat different from other

cytotoxic agents: even though the reactive bleomycin intermediate interacts with DNA, it

is the oxidative damage that triggers cell death but not the bleomycin–DNA complex

itself. In one in vitro study, after degradation of DNA treated with activated bleomycin,

the products included oligomers terminated by 5′-phosphate [51] and nucleobase

fragments containing deoxyribose carbons 1′–3′ [52], which were identified as base

propenals: 3-(pyrimidin-1-yl)-2-propenal and 3-(purin-9-yl)-2-propenal (Figure 12) [48].

17

Figure 12. DNA degradation products after bleomycin-induced strand cleavage. The final products are stable and have been isolated.

1.2.2 Targeted Therapies

While there is continued interest in the discovery of improved cytotoxic agents,

molecular and genetic methods of studying cell biology unveiled entirely new pathways

involved in cancer cell proliferation and survival. Researchers set out to study the

molecular defects in cancer cells and how to target them without harming normal cells [7].

The new targets include growth factors, signaling molecules, cell-cycle proteins,

modulators of apoptosis, and molecules controlling angiogenesis [53].

A highlight in the design of targeted anticancer therapy is the development of

imatinib mesylate (Glivec), which was derived from a natural product by Novartis. It is a

moderately potent inhibitor of the kinase BCR–ABL, the fusion protein product of a

chromosomal translocation that is involved in the pathgenesis of chronic myeloid

leukemia (CML) [7]. Another class of compounds was developed to inhibit the epidermal

growth factor receptor (EGFR). Gefitinib, for instance, is a competitive inhibitor of

EGFR, which causes partial remissions in 10–15% of patients affected with NSCLC [54].

Although the discovery of molecular targets and pathways specific to cancer cells

is becoming more and more important, it remains uncertain if targeted agents will ever

replace existing cytotoxic chemotherapies. Current targeted agents are only beneficial if

co-administered in tandem with cytotoxic agents [55]. For example, Avastin

O BPO

OP

(i) O2(ii) e-, H+

O BPO

OP

OHODNA cleavage

OO

PO B

OOP

4'3'

4'

3'

4'

3'

18

(bevacizumab, vascular endothelial growth factor inhibitor, Genentech/Roche) shows

improved responses in colorectal cancer patients co-treated with oxaliplatin (trans-1,2-

diaminocyclohexaneoxalatoplatinum(II)), but has demonstrated no activity as a single

agent [55].

1.3 Platinum Antitumor Agents

1.3.1 Bifunctional Platinum Crosslinkers

In 1845, the platinum complex cis-diamminedichloroplatinum(II), now known as

the antitumor agent cisplatin, was first reported by M. Peyrone [14]. Little research was

done on this complex until Rosenberg rediscovered it in the 1960s when he found that E.

coli cells cannot divide when exposed to it [56]. Detailed chemical analysis identified two

active complexes―the neutral cis-complex cisplatin and a platinum(IV) analogue―as

the cause of this intriguing biological effect. In 1968, cisplatin was administered

intraperitoneally to mice bearing a xenografted tumor at the non-lethal dose of 8 mg/kg

and was shown to cause marked tumor size reduction [57].

Inspired by these findings, the first patients were treated with this agent only three

years later in 1971, a remarkably short time period, in modern terms, from the original

bench discovery to the clinic. Cisplatin underwent extensive clinical trials afterwards and

was approved by the US Food and Drug Administration (FDA) in 1978. Since then,

cisplatin has revolutionized the treatment of certain cancers, especially malignancies of

the urogenital tract [56].

There is strong evidence to support the view that the mechanism of action of

cisplatin (Figure 13) [58] involves intracellular activation by aquation of one of the two

19

chloride leaving groups and subsequent adduct formation with nuclear DNA.

Unfortunately, the activated platinum intermediate also exhibits a high affinity for amino

acid sulfur and binds to proteins and peptides containing cysteine and methionine. These

include the tripeptide glutathione (GSH) and metallothioneins, binding to which

contributes to the toxic side effect of cisplatin and also causes the drug’s detoxification,

which may lead to tumor resistance. Cisplatin–DNA adducts activate various signal-

transduction pathways involved in DNA-damage recognition and repair (nucleotide

excision repair pathway), cell-cycle arrest, and cell death/apoptosis [59].

Figure 13. Mechanism of action of cisplatin. Taken with permission from reference [58].

20

Figure 13 shows how platinum enters cells by either trans-membrane transporters

(like the copper transporter CTR1), or by passive diffusion [58]. Once inside the cell,

cisplatin is activated by reaction with water to form chemically reactive aqua species.

This step is facilitated by the relatively low chloride concentration in the cytosol. The

activated intermediate binds to DNA to form DNA adducts. In some platinum-resistant

cancer cells, GSH and metallothionein levels are relatively high, so the activated

intermediate is irreversibly trapped before DNA binding can occur, which causes drug

resistance. Platinum drugs are exported from the cells by the glutathione-S-conjugate

export pump as well as other transporters, which also contributes to platinum drug

resistance [58].

Figure 14. Binding modes between cisplatin and DNA (intrastrand and interstrand cross-links). Taken with permission from reference [58].

Early studies [58] conducted in vitro with salmon sperm DNA exposed to

cisplatin showed platinum binding to the N7 position of the imidazole ring of the purine

bases of guanine (G), and to a lesser extent, adenine (A), to form either monofunctional

(loss of one leaving group) or bifunctional adducts (loss of both leaving groups) (Figure

PtH3N Cl

H3N OH2

NN

N

NO

N

NN

N

O

HH

H

HH

Guanine N7 position

Intrastrand cross-link (major)

Intrastrand cross-link (minor)

PtNH3

NH3

PtNH3

NH3

21

14) [60]. The majority of adducts are formed on the same DNA strand and involve bases

adjacent to one another, and are therefore known as intrastrand adducts or cross-links.

These include 1,2-GpG intrastrand (60%–65%) and 1,2-ApG intrastrand (20%–25%)

adducts [61]. The less frequently formed adducts are the 1,3-GpXpG intrastrand cross-

link [62] and G–G interstrand cross-link on opposite DNA strands of the double helix

[63]. All these structures have been elucidated either by X-ray crystallography or by

solution structures solved using NMR spectroscopy [61-63].

Even though cisplatin is a very effective anticancer drug, it is notorious for its

toxicity to the kidneys (nephrotoxicity) and gastrointestinal tract [64]. In addition to its

systemic toxicity, other disadvantages of cisplatin are its (i) limited applicability in a

narrow range of tumors, (ii) inactivation by increased levels of thiol-containing proteins

and peptides, such as GSH, and enhanced DNA repair (iii) low water solubility, (iv)

inefficient delivery to the tumors, and (v) inconvenient administration (cisplatin cannot

be taken via oral route). Therefore, three major strategies with the purpose of

circumventing these drawbacks have been proposed: first, the design of improved

platinum drugs; second, to enhance delivery of platinum to tumors; and third, to combine

platinum drugs with new molecularly targeted drugs. By following these strategies,

second- and third- generation cisplatin analogues have been developed, and a platinum

drug “family tree” has been generated (Figure 15) [58].

Carboplatin (cis-diammine-[1,1-cyclobutanedicarboxylato]platinum(II)) was

developed and introduced into the clinic in the mid-1980s [65] as a second-generation

derivative of cisplatin. Its design is based on the idea that a more stable leaving group

other than chloride might decrease the toxicity without affecting antitumor efficacy.

22

Compared with cisplatin, carboplatin largely eliminates nephrotoxicity and causes less

neurotoxicity. The adducts formed by carboplatin on DNA are essentially the same as

those formed by cisplatin, but 20–40-fold higher concentrations of carboplatin are

required to achieve the same therapeutic effect, and the rate of adduct formation is about

an order of magnitude slower [66].

Figure 15. The current platinum drug “family tree” and generalized rationale underlying their development. The dates indicate when each drug was first given to patients.

PtH3N Cl

H3N Cl

PtH3N O

H3N O

O

O

Pt

H2N O

NH2

O

O

O

PtH3N Cl

NH2

Cl

O

O

O

O

PtH3N Cl

N Cl

Pt

H2N O

NH2

O

O

O

RR

Pt NH2

O

H2NNH

O

O

O

ONH

OHN

ONH

O

O

HN

OH

n

Cisplatin (1971)

Improved safety

Broader spectrum ofantitumor activity

Carboplatin (1982)

Improved patientconvenience(oral activity)

Broader spectrumof activity; retentionagainst acquired resistance to cisplatin

Oxaliplatin (1986)

Improved delivery

Satraplatin (JM216) (1993)Picoplatin(JM473) (1997)

NaR = C9H19

DACH-L-NDDP(Aroplatin) (2004)

AP5346 (ProLindac) (2004)

Cisplatin (1971), 14

Carboplatin (1982), 15

Oxaliplatin (1986), 16

Satraplatin (JM216) (1993), 17Picoplatin, (JM473) (1997), 18

DACH-L-NDDP (Aroplatin), (2004), 19 AP5346 (ProLindac) (2004), 20

23

Oxaliplatin (1R,2R-diaminocyclohexaneoxalatoplatinum(II)) was designed on the

basis of the bulky 1,2-diaminocyclohexane (DACH) ligand and went into clinical trails in

the early 1990s [67]. Interestingly, unlike for cisplatin and carboplatin, the cellular

accumulation of oxaliplatin seems to be less dependent on the copper transporters [68].

There are also several differences between the structures of cisplatin-induced and

oxaliplatin-induced 1,2-intrastrand DNA cross-links [69], and oxaliplatin retains activity

against some cancer cells with acquired resistance to cisplatin [70].

Satraplatin (bis-acetatoamminedichlorocyclohexylamineplatinum(IV), JM216)

was initially developed as an orally active version of carboplatin. Preclinical studies

showed that the drug possesses good oral antitumor activity, comparable to that of

intravenously administered cisplatin or carboplatin in human ovarian cancer xenografted

into mice [71]. Picoplatin (cis-amminedichloro-2-methylpyridineplatinum(II); JM473)

was rationally designed to provide steric hindrance around the platinum center [72].

Picoplatin reacts less avidly with thiol-containing species, such as GSH, than cisplatin.

The drug shows good activity against a wide range of cisplatin-resistant cells in vitro [72]

and possesses antitumor activity by both intravenous and oral routes in vivo [73]. DACH-

L-NDDP (Aroplatin) and AP5346 (ProLindac) are two newly developed platinum drug

formulations, aimed at improving drug delivery. Aroplatin is based on liposomal

preparations of cisplatin-like molecules [74], and ProLindac is synthesized by combining

a platinum-based drug with a water-soluble, biocompatible co-polymer [75]. Both

formulations have entered clinic trials against cisplatin-resistant tumors.

Even though thousands of platinum compounds have been evaluated for their

anticancer activities with varied success, the majority obey the classical structure–activity

24

relationships (SAR) for cisplatin-type complexes, summarized by Cleare and Hoeschele

[76]. These relationships state that a Pt(II) complex does not show antitumor activity

unless the Pt(II) complex exhibits a cis geometry, cis-[PtX2(Am)2], where X stands for a

good leaving group and Am is an inert amine with at least one NH moiety. Cisplatin-type

drugs show similar toxicity profiles, form the same type of cross-links, and share the

same spectrum of bioactivity [76].

Therefore, major efforts have been made to design compounds that act unlike

traditional cisplatin-type drugs, with the common goal of discovering agents that offer

substantial clinical advantages over the existing drugs and target cancers that previously

did not respond to cisplatin-type drugs. A number of researchers have taken different

approaches to design Pt drugs based on an improved understanding of the mechanisms of

drug resistance. Several of the recent approaches will be discussed in the following

sections.

1.3.2 Monofunctional Platinum Complexes

In a search for new drugs that exhibit biological activity complementary to that of

cisplatin, several new classes of platinum complexes possessing unique geometries and

ligand sets have been developed. Their design is based on the supposition that such non-

classical complexes might act through a mechanism of action distinct from that of

cisplatin and exhibit improved pharmacological properties. These new platinum

complexes include monofunctional platinum complexes, trans-complexes, multinuclear

platinum agents, as well as platinum–intercalator conjugates.

25

Several monofunctional platinum(II) complexes showed promise as a new type of

antitumor agent that does not follow the Cleare–Hoeschele SAR [77, 78]. The general

formula for monofunctional platinum complexes is [PtL3Cl], in which Cl- acts as the only

leaving group.

Figure 16. Examples of bioactive monofunctional platinum complexes.

Figure 16 shows examples of bulky planar ligands such as quinolines [77] that

have been introduced as new non-leaving groups in non-classical platinum anticancer

agents. These ligands significantly lower the rates of platinum binding to sulfur-

containing biomolecules [79]. On the other hand, the binding mode of these complexes

with DNA appears to be radically different from that of existing platinum drugs, and their

spectrum of activity is different from the established pattern of cisplatin. It has been

reported that monofunctional platinum(II) complexes significantly destabilize DNA and

modify the conformation of DNA, similar to the cross-linking agents [78].

1.3.3 Other Non-classical Platinum Agents

The trans isomer of [PtCl2(NH3)2], known as transplatin, lacks chemotherapeutic

activity [80]. Unlike cisplatin, which forms 1,2-intrastrand crosslinks, geometric

constraints prevent transplatin from forming this adduct. Instead, 1,3-intrastrand cross-

Pt NH2

Cl

N

O

NPt NH2

Cl

N

O

N N

N

PtCl

S

O HN

O

O

21 22 23

26

links are formed [81]. Interstrand cross-links of transplatin are formed between G and C

residues of the same base pair as shown in Figure 17 [80], which highlights the markedly

different DNA conformations induced by the two platinum isomers [61, 82].

In addition, transplatin reacts significantly faster with GSH than cisplatin, because

of the mutual trans-labilization of the two chloro ligands in the molecule, which causes

rapid cellular detoxicification of this isomer. However, several derivatives of trans-

platinum complexes have been described in the literature that show improved activity.

These include analogues containing sterically hindered planar nitrogen-containing

heterocycles (pyridine, quinoline, etc). These compounds show reduced reactivity with

GSH and form cytotoxic monofunctional DNA adducts [83].

Figure 17. Interstrand cross-links for cisplatin (left) and transplatin (right). Taken with permission from reference [80].

Multinuclear platinum complexes represent another class of compounds that have

shown great potential in cancer chemotherapy [77]. These complexes usually contain two,

three or four platinum centers with cis and trans configurations and bind to DNA in a

pattern different from that of cisplatin. Until recently, over 50 multi-nuclear platinum

27

complexes have been synthesized and tested for cytotoxicity in a variety of cancer cell

lines [84]. Most of these complexes react with DNA more rapidly than cisplatin and

produce long-range interstrand and intrastrand cross-links [85].

From all the complexes which have been tested, the first and most important

multinuclear platinum complex to enter phase II clinical trials based on its excellent

performance in vitro and in vivo was the trinuclear complex BBR3464 (Figure 18) [86].

BBR3464 is significantly more active than cisplatin in all of the cancer cell lines tested,

including pancreatic, lung, and skin cancers. It shows activity comparable to cisplatin at

up to 500-fold lower doses in vitro [87, 88]. In addition, BBR3464 is also capable of

overcoming cisplatin resistance and shows activity in several cancer cell lines that have a

natural resistance to cisplatin [87].

Figure 18. Structure of the trinuclear platinum complex BBR3464.

The high positive charge and amine groups of BBR3464 are believed to

contribute to the recognition of target sites on DNA through electrostatic and hydrogen-

bonding interactions [89]. The rapid binding to BBR3464 leads to various long-range

interstrand and intrastrand cross-links, where the interstrand adducts account for about

20% of the DNA adducts [84].

BBR3464, 24

PtH3N

NH3

H2N

ClNH2

Pt

H2NH3N

NH3

NH2

Pt ClH3N

NH3

4+

28

1.3.4 Platinum–Intercalator Conjugates

In platinum–intercalator conjugates, a polycyclic planar (usually fused, and

positively charged, aromatic rings) group is tethered to a divalent platinum center through

one or multiple thermodynamically stable donor–metal bond(s) that is (are) not reversed

under physiological conditions or upon binding of one of the two components to DNA.

Therefore, platinum center and intercalator are functionally independent. Platinum bears

at least one substitution-labile group that can be replaced with DNA nucleobase nitrogen

to form a stable coordinative bond. The linker group that connects the organic and

inorganic portions of the conjugate should be somewhat flexible, like polymethylene

chains, to facilitate a hybrid binding mode [90]. A schematic drawing summarizing

various functionalities is shown in Figure 19 [90].

Figure 19. Schematic drawing of a platinum–intercalator conjugate. Adopted from reference [91] with modifications.

The rational design of conjugates combining platination and intercalation was

inspired by the early studies of the DNA binding of cisplatin in the presence of

intercalating agents using molecular biology and biophysical methods [91, 92]. These

include cisplatin complexes conjugated to both non-therapeutic classical intercalators,

such as acridine orange [93, 94] and clinical agents, such as the topoisomerase poison

DNA intercalator

flexible linkerchain Substitution-

inert groupSubstitution-labile group

Square-planarplatinum center

29

doxorubicin (Figure 20) [95, 96]. The platinum–doxorubicin conjugate shows activity in

various cancer models in vivo, such as murine leukemias (L1210, P388), which are

resistant to both cisplatin and doxorubicin. In the solid tumor cell lines, the overall

activity of this conjugate resembled that of doxorubicin more than that of cisplatin [96].

As a continuation of the biophysical studies, a series of monofunctional and

bifunctional platinum–intercalator conjugates, including platinum–ethidium and

platinum–9-aminoacridine, have been developed (Figure 20).

Figure 20. Structures of platinum–intercalator conjugates.

N

NHPt

Cl NH2

Cl

Me2N NMe2

Platinum-Acridine Orange Conjugate

O

O

OH

OHMeO O

O OH

OH

O

OH H2NPt

BuH2N

Cl

Cl

Platinum-Doxorubicin Conjugate

N

NH2Pt

Cl

H3N NH3

H2N

2+

Platinum-Ethidium Conjugate

NH

HNPt

H3N

Cl

NH3

+

Platinum-9-Aminoacridine Conjugate

N

HN

R R

(CH2)n

N

HN

(CH2)nNH

H2N

NH2

PtCl

Cl PtNH2

Cl Cl

Platinum-9-Anilinoacridine Conjugates

Platinum-Acridine Orange Conjugate, 25 Platinum-Doxorubicin Conjugate, 26

Platinum-Ethidium Conjugate, 27 Platinum-9-Aminoacridine Conjugate, 28

Platinum-9-Anilinoacridine Conjugates, 29 and 30

30

Another design rationale is based on the hypothesis that the tethering of cisplatin

to DNA-affinic ligands might have favorable pharmacokinetics by reducing the residence

time in the cytosol and improving the DNA binding kinetics of the drug. Two types of

platinum–anilinoacridine conjugates (Figure 20) have been synthesized that were able to

overcome cross-resistance to cisplatin in vitro. Disappointingly, the compounds’ activity

in vitro did not translate into anti-tumor activity in vivo, probably due to their poor

solubility [97].

1.4 Background and Significance of the Doctoral Research Program

1.4.1 The Development of PT-ACRAMTU

The development of cisplatin–acridine complexes was inspired by the known

topoisomerase II poisoning activity of the free 9-aminoacridines and their increased water

solubility compared to the aniline derivatives [90]. In addition, it was demonstrated that

the DNA-adduct profile of cisplatin can be modulated by an acridine intercalator [98, 99].

Our laboratory has taken a more radical approach of preventing the metal from cross-link

formation of consecutive guanines in the major groove by tethering a monofunctional

platinum moiety to a minor groove directed intercalating agent, 1-[2-(acridin-9-

ylamino)ethyl]-1,3-dimethylthiourea (ACRAMTU, 37) [100].

The rationale behind PT-ACRAMTU ([PtCl(en)(ACRAMTU)](NO3)2, en = ethane-

1,2-diamine, ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea,

acridinium cation) (31) was to design a platinum complex that contains one labile group

that can be substituted by nucleobase nitrogen to form a coordinative bond, and an

31

intercalator attached to the platinum through a flexible linker, which can stack between

adjacent DNA base pairs.

Figure 21. Design of PT-ACRAMTU (31).

As shown in Figure 21, there are several significant features [90] of the dual binding

mode agent PT-ACRAMTU distinguishing it from cisplatin and its second- and third-

generation derivatives: 1) A single chloride leaving group can be expected to favor

monofunctional binding of the metal with nucleobase nitrogen in double-stranded DNA.

2) The idea of introducing a Pt–S bond is based on the strong affinity of sulfur for

platinum, which ensures ACRAMTU remains attached to platinum as a non-leaving

group. 3) A semirigid linker was chosen to optimize strainless intercalation and

platination with minimum spacing between them to favor platination of DNA bases

directly adjacent to the intercalation sites. 4) Acridine as a typical intercalator helps the

complex pre-associate with DNA via π–π stacking with base pairs and facilitates the

coordinative binding of platinum to nucleophilic sites of purines. 5) The 2+ charge

enhances electrostatic attraction between the drug and negatively charged DNA. 6) The

PT-ACRAMTU, 31

PtH2N NH2

Cl S

NH

N NH

NH

bidentate non-leaving group

2+

chloro leaving group

Pt-S linkage

semirigid ethylene-thiourealinker unit

9-aminoacridinium,a classical intercalating unit

dicationic character

32

chelating nonleaving group ethane-1,2-diamine (ethylenediamine, en) mimics the

cisplatin structure and enhances the platinum moiety’s stability.

1.4.2 Synthesis of PT-ACRAMTU

Figure 22. Synthesis of PT-ACRAMTU (31).

The synthesis of PT-ACRAMTU (31) shown in Figure 22 utilizes the activated form

of enplatin ([PtCl2(en)]), [PtCl(en)(DMF)]+, which is reacted with the acridinylthiourea

nitrate salt, ACRAMTU·HNO3 (37·HNO3), in dry DMF. The synthesis of 37 nitrate salt

involves: a) selective protection of the secondary amine nitrogen in 32 with tert-

butylcarbamate, BOC, b) nucleophilic substitution of 9-phenoxyacridine with protected

amine 34 [101], c) deprotection to remove the Boc group, and d) addition of

H2NHN

1.CF3COOEt/THF/ <10 oC

2.(Boc)2O/THF/<10 oC NH

NF3C

OO

O

0.2M NaOH/48h, 35oCH2N N O

O

1.HCl, CH3COOH 1.MeSCN/EtOH/Δ

enplatin/AgNO3

N

O

NHN N

O

ON

HN N

H

HNHN N N

H

S

+

HNHN N N

H

S Pt ClH2N NH2

2+

THF/Δ 2.HNO3

DMF/dark/14h

2.Base

32 33 34

35 36

37·H+ 31

33

methylisothiocyanate to form 37, followed by protonation with one equivalent of nitric

acid to form the desired acridinium salt [102, 103].

1.4.3 Biological Activity and DNA Binding Mechanism

PT-ACRAMTU (31) has shown good biological activity in cell proliferation

assays. In several cell lines, PT-ACRAMTU proved to be more active than cisplatin and

the Pt-free ligand ACRAMTU itself. These include HL-60 leukemia, as well as solid

tumors, such as NSCLC (Table 1) [104].

Table 1. Cytotoxicity of PT-ACRAMTU (31) in NSCLC.

aIC50 values are concentrations of drug required to inhibit colony growth by 50% compared to controls and represent averages of at least two experiments plus or minus the standard error of the mean. Data adopted from reference [105].

An unusual observation was made when performing enzymatic digestion of calf

thymus DNA treated with PT-ACRAMTU. Four platinated DNA fragments were isolated

and characterized by HPLC–MS and X-ray crystallography, some of which were entirely

unexpected. While the majority of adducts are formed with N7 of guanine (major groove,

80%), the three other platinum-modified DNA adducts proved to be quite unusual. PT-

ACRAMTU forms adducts with adenine at three sites [105], N7 (~11%), N3 (~7%), and

N1 (~2%). It can be said that the seemingly simple replacement of one chloride leaving

group with a classical intercalator (ACRAMTU) in [PtCl2(en)], results in a dramatic

decrease in the formation of guanine adducts.

Cell line A549 Calu-1 SK-LU-1 H358 H460 H522 H596

IC50 (μM) a 0.23±0.05 1.41±0.72 0.26±0.06 0.50±0.20 0.17±0.02 0.095±0.02 0.18±0.03

34

Figure 23. Potential reaction pathways of Pt–guanine adduct formation.

The binding mechanism of PT-ACRAMTU (31) in duplex DNA differs from that

of cisplatin. It does not require an activation step before it associates with DNA because

of its positive charge, whereas cisplatin has to form a cationic complex via hydrolysis,

which enables electrostatic association of the drug with nuclear DNA. A major effort has

been devoted to elucidating the DNA-binding mechanism of PT-ACRAMTU utilizing

various techniques, including analytical (HPLC–MS) [105, 106], biochemical (enzymatic

digestion and footprinting assays) [107, 108] and NMR experiments (NMR solution

structure, 1H and 2D [1H–15N] heteronuclear single quantum coherence (HSQC) kinetic

NMR studies) [109, 110] (Figure 23): The formation of the guanine N-7 adduct by PT-

ACRAMTU (31) is neither a simple first-order nor second-order process. The first step

involves rapid intercalation from the minor groove. In the subsequent step, nucleophilic

SPt

N N

OH2

3+

ACR

SPt

N N

N7-dG

3+

ACR

+ H2O

+ Cl-

+ dG+ dG

k1

k-1

k2direct pathway??

dG = 2'-deoxyguanosine

SPt

N N

Cl

2+

ACRSPt

N N

Cl

2+

ACRintercalation

SPt

N N

OH

2+

ACR

?

35

substitution of the chloride leaving group involves two possible pathways: reversible

hydrolysis followed by replacement of aqua ligand by nucleobase nitrogen or direct

replacement of chloride by nucleobase, which might be favored by the cis-effect of

thiourea sulfur [111]. Based on the data acquired in the previous study, the latter pathway

seems a possibility.

1.4.4 Goals of Doctoral Research Program

The overarching goal of the research presented in this dissertation was to study

structure–activity relationships (SAR) in platinum–acridine hybrid antitumor agents. To

achieve this goal, the current work implemented a classical medicinal chemistry approach

involving synthesis, mechanistic studies at the target level, and biological evaluation.

The work was motivated by the discovery of the DNA-targeted anticancer agent PT-

ACRAMTU (31), which has DNA-binding properties different from those observed for

clinical platinum anticancer agents. The design of the compound does not follow the

Cleare–Hoeschele SAR, and it is believed that its cytotoxic properties are mediated by

the formation of DNA adducts different from those induced by cisplatin and other

platinum anticancer agents. PT-ACRAMTU is the first platinum-based anticancer agent

that forms unusual adenine adducts in the minor groove, which may contribute to its

biological activity.

While the cytotoxicity data previously acquired for PT-ACRAMTU are promising,

the agent proves to be only slightly more cytotoxic than cisplatin and, disappointingly,

was found to be inactive in vivo (unpublished data). In addition, the sulfur coordination

in the prototype was found to give rise to a high reactivity of the metal which leads to

36

slow degradation of the complex in buffered solution. Therefore, a systematic study of

chemically more stable derivatives of this compound seems warranted in order to develop

a new conjugate with enhanced activity in cancer cells and antitumor activity in vivo.

Based on the structural and thermodynamic requirements for the recognition and

removal of DNA lesions by the NER complex, the monoadducts formed by PT-

ACRAMTU can be expected to be less efficiently repaired than cisplatin–DNA cross-

links because they do not bend or destabilize the DNA helix. PT-ACRAMTU has the

ideal geometry and conformational flexibility to allow a binding mode that combines

nucleobase platination and strainless classical intercalation perpendicular to the helical

axis. It is therefore plausible to assume that retaining the previously optimized core

structure of PT-ACRAMTU in the new set of compounds is an appropriate strategy for

identifying a potent analogue whose adducts are able to circumvent NER. Thus, one goal

pursued in Chapters 2 and 3 was to generate nitrogen-donor-based mimics of

ACRAMTU, based on guanidine- and amidine-modified 9-aminoacridine, and to

introduce them as carrier ligands in the corresponding conjugates. The idea behind this

approach was that tuning of the metal’s reactivity by replacing the thiourea-S in

ACRAMTU with an imino-NH donor might have an effect on the conjugate’s target

affinity and selectivity without altering the structure of its cytotoxic DNA adducts. To

achieve this, new organic synthetic methodology and conjugation chemistry were

developed. To establish relationships between chemical structure and biological activity,

the reactivity of the newly designed compounds with biomolecular targets was studied in

model systems using NMR and UV-visible spectroscopy and in-line liquid

chromatography/mass spectrometry. The mechanistic work was complemented by

37

experiments addressing the cytotoxic activity and the subcellular and tissue distribution

of the compounds.

In another approach, a platinum–acridine conjugate was developed in which the

linkage geometry (the point of attachment of the platinated side chain) deviated from that

in the prototype, PT-ACRAMTU. While in the latter structure platinum is linked to the

9-position of the acridine chromophore, the new derivative contains a 4-carboxamide

chain. Since the 9- and 4-positions are located on opposite sides of the aromatic system,

it was speculated that the directionality of intercalation might affect the DNA sequence

and groove specificity of the platinum moiety, leading to an altered DNA adduct profile

and, potentially, an altered cellular response. The results of this study, which include

synthesis and characterization of the target compounds, DNA binding studies of the

carrier ligands, and growth inhibition assays in cancer cell lines, are presented in Chapter

4.

38

CHAPTER 2

SYNTHESIS, CHARACTERIZATION, AND BIOLOGICAL EVALUATION OF

GUANIDINE DERIVATIVES OF ACRAMTU

The work contained in this chapter was initially published in Journal of Organic

Chemistry in 2007. The manuscript, including figures and schemes, was drafted by

Zhidong Ma and Dr. U. Bierbach and edited by Dr. U. Bierbach before submission to the

journal. Since the publication, changes in both format and content have been made to

maintain a consistent format throughout this work. The experiments described were

performed by Zhidong Ma, except for the crystal structure determinations, which were

contributed by Dr. C. S. Day, and the cytotoxicity assays which were contributed by Dr.

G. L. Kucera and G. Saluta.

39

2.1 Background

Acridine derivatives have a broad range of applications as chemotherapeutic agents in

the treatment of cancer. They show a high affinity for DNA and rapid association rates

with this biomolecule. The geometry of the fused aromatic ring systems have

approximately the dimension of DNA base pairs with which they tend to π-stack [13, 112,

113]. Thus, the primary cellular target of acridines is genomic DNA, to which they bind

through intercalation to inhibit important DNA-associated enzymes, such as

topoisomerases [113]. The biological activity of these agents is mainly determined by

two factors, namely the DNA-binding affinity and the dynamic properties of the

intercalator–DNA complex. These two factors can be optimized by tuning the electronics

of the acridine chromophore (which also affects its protonation state), for instance by

introducing pendant, non-intercalating groups [114].

PT-ACRAMTU (31), derived from 1-[2-(acridine-9-ylamino)ethyl]-1-3-

dimethylthiourea (ACRAMTU, 37), has been developed to combat the notorious

resistance of certain tumors to current DNA-targeted therapies (Figure 24) [90, 100].

X = S, thiourea derivative, “ACRAMTU”, 37 X = NH, guanidine derivative, “ACRAMGU”, 38

Figure 24. Structure of ACRAMTU and its guanidine analogue.

HN

HN

N NH

X

40

While the protonated form (pKa ≈ 9.8) of ACRAMTU, which intercalates into DNA

with the thiourea moiety residing in the minor groove, proved to be moderately cytotoxic

to certain cancer cells [109], it produces its cytotoxic potential as a DNA-targeted carrier

ligand in platinum–intercalator hybrid agents, in which thiourea sulfur is modified with a

DNA–metalating group [10, 100]. Several second-generation derivatives of PT-

ACRAMTU have been synthesized, which demonstrate excellent activity in

chemoresistant non-small cell lung cancers in vitro (Table 1) [10].

Although some platinum–thiourea conjugates demonstrated remarkable biological

activity in vitro, they seem to lack a true advantage over PT-ACRAMTU, whose

cytotoxicity did not translate into the anticipated antitumor activity against cancers in

vivo. In addition, the platinum–Sthiourea bond is quite reactive, which causes ligand

scrambling in PT-ACRAMTU (31) in aqueous solution. The extra lone pair of electrons

on sulfur is able to replace the leaving group (chloride) on another PT-ACRAMTU

molecule which leads to slow decomposition of the drug. The resulting

bis(acridinylthiourea)platinum(II) complex (39) [115] can be isolated from aged solutions

of the drug prototype (Figure 25). While compound 39 itself is a cytotoxic agent that

strongly binds to native DNA because of its bisintercalative binding mode [115], it is an

undesired impurity in preparations of PT-ACRAMTU. It was, therefore, deemed

necessary to replace the platinum–thiourea bond with a less reactive linkage.

41

Figure 25. Slow decomposition of PT-ACRAMTU due to nucleophilicity of coordinated thiourea sulfur.

Another reason that prompted us to make changes to the platinum–acridine

linkage was to explore ways of modulating the kinetics of DNA adduct formation. In

Chapter 1, (Section 1.4.3), we have speculated that the cis-effect of thiourea sulfur may

play an important role in the irreversible binding step of the drug, the step in which

chloride on the metal center is substituted by nucleobase nitrogen. It has been

demonstrated by Tobe and his group (Figure 26) that for the complexes

[PtCl(en)(DMSO-S)]+ and [PtCl(en)(DMS)]+ (DMSO = dimethylsulfoxide, DMS =

dimethylsulfide), a sulfur donor greatly accelerates chloride substitution by an incoming

nucleophile over analogous complexes with a nitrogen donor set. While the relative rate

enhancement (kS/kN) was found to be large for strong incoming nucleophiles, such as

sulfur ligands (SCN-), it is relatively small for weakly nucleophilic ligands, such as H2O

[116, 117].

HN

HN

N

S

NH

PtNH2H2N

Cl

2+

HN

HN

N

S

NH

PtNH2H2N

Cl

2+

HN

HN

N

S

NH

PtNH2H2N

4+

NH

HN

N

S

HN

+

buffer, mediaweeks

"[Pt(en)Cl2]"aquated species

39

31 31

42

Figure 26. Mechanistic implications of the kinetic cis-effect. Data adopted from references [116] and [117].

Thus, to improve the stability of the conjugate and to investigate the possibility of

kinetic tuning of the Pt center, we proposed to replace thiourea sulfur (sp2) in

ACRAMTU with imino nitrogen (sp2). Nitrogen is a donor group low in the cis-influence

series, which also lacks, unlike sulfur, the reactive lone pair electrons when attached to

the metal. The core structure of the prototype will be virtually unchanged in such a

derivative (Figure 24). The expectation would be that platinum and imino nitrogen would

form a thermodynamically stable bond, such as in analogous iminoether-substituted

(HN=C(OR)R’) complexes, which have been tested for antitumor activity [118]. We

were particularly interested in generating a guanidine analogue of ACRAMTU, in which

thiourea sulfur has been replaced with imino nitrogen (Figure 24). The synthetic studies

leading to this molecule, 1-[2-(acridine-9-ylamino)ethyl]-1,3-dimethylguanidine

(ACRAMGU, 38), which reveal unexpected reactivity features of the 9-aminoacridine

chromophore, are summarized in this chapter.

Pt

H2N

NH2

Cl

L

++ Pt

H2N

NH2

Y

L

(2-n)+

Y

H2O

NH3

SCN-

Relative 2nd Order Rates

+ ClYn-

L = NH3 L= DMSO-S

1 1.1

1000 10000

15000 250000

43

2.2 Experimental Section

2.2.1 Materials and Chemicals

1H NMR spectra of the target compounds and intermediates were recorded on

Bruker Advance 300 and DRX-500 instruments operating at 500 and 300 MHz,

respectively. 13C NMR spectra were recorded on a Bruker Advance 300 instrument

operating at 75.5 MHz. Chemical shifts (δ) are given in parts per million (ppm) relative to

internal standards trimethylsilane (TMS), or 3-(trimethylsilyl)-1-propanesulfonic acid

sodium salt (DSS) for samples in D2O. 1H and 13C NMR spectra for the new compounds

(38, 40, 42, 43, 45, 46, and 47) are available in Appendix A. All the target compounds

(38, 40, 42, and 43) were fully characterized by gradient COSY and 1H-detected gradient

HMQC and HMBC spectra recorded on a Bruker DRX-500 MHz spectrometer.

Elemental analyses were performed by Quantitative Technologies Inc., Madison, NJ. All

reagents were used as obtained from commercial sources without further purification

unless indicated otherwise. Solvents were dried and distilled prior to use. Flash

chromatography was performed using ACROS (50-200 mesh) neutral alumina.

The following compounds were prepared according to preciously described

procedures: N-acridin-9-yl-N'-methylethane-1,2-diamine (36) [100], 1-(2-(acridin-9-

ylamino)ethyl)-1,3-dimethylthiourea (ACRAMTU, 37) [100], and di(imidazol-1-

yl)methanimine (41) [119].

2.2.2 Synthesis and Characterization of Guanidine Derivatives

(Z)-N-[1-(Acridin-9-yl)-3-methylimidazolidin-2-ylidene]methanaminium

Hydrochloride, 40. To a solution of 1-[2-(acridin-9-ylamino)ethyl]-1,3-

44

dimethylthiourea (ACRAMTU, 37, 127 mg, 0.39 mmol) in 5 mL dry tetrahydrofuran

(THF) was added dropwise phosgene in toluene (1.41 mL, 1.93 M solution) at 0 oC. The

mixture was stirred for 2 h at this temperature and for another 1 h at room temperature.

The solvents and excess of phosgene were removed in a vacuum. The hygroscopic

residue was dissolved in 120 mL of dry acetonitrile, and into this solution was bubbled

ammonia gas for 2 h. Solvent was evaporated, and the residue was recrystallized from

ethanol to afford the product as yellow needles. Yield: 98 mg (76%); mp 238 – 239.5 oC.

1H NMR (D2O): δ 8.09 (4H, two overlapping d), 7.92 (2H, t, J = 8.7 Hz), 7.78

(2H, t, J = 7.5 Hz), 4.17 (H7a and H8a, 4H, m), 3.27 (H6a, 3H, s), 1.98 (H4a, 3H, s).

13C-{H} NMR (D2O): δ 158.2, 148.8, 140.1, 132.26, 129.0, 128.6, 123.6, 122.3,

51.6, 49.8, 32.9, 29.1.

ESI-MS (MeOH, +ve mode) m/z: 291.9 [M]+.

Anal. (C18H19N4Cl·0.5C2H5OH) C, H, N: calcd, 65.23, 6.34, 16.01; found, 65.52,

6.19, 15.77.

2′-(1H-Imidazol-1-yl)-8′-methyl-7′,8′-dihydro-6′H,10H-spiro-[acridine-9,4′-

imidazo[1,2-a][1,3,5]triazine], 42. To precursor 36 (251 mg, 1.0 mmol) in 10 mL of dry

THF was added di(imidazol-1-yl)methanimine (41) (338 mg, 2.1 mmol). The mixture

was allowed to stir at room temperature for 10 h, and then stored at - 4 oC for several

days. The resulting yellow crystals were filtered off, washed with cold THF and ether,

and dried under vacuum. Yield: 173 mg (47%); mp 261.5 – 262.5 oC.

1H NMR (DMF-d7): δ 9.58 (NH, 1H, s), 8.40 (H11a, 1H, t), 7.71 (H13a, 1H, t, J =

1.1 Hz), 7.42 (H1 and H8, 2H, d, J = 7.9 Hz), 7.26 (H3 and H6, 2H, t, J = 7.6 Hz), 7.04

45

(H4 and H5, 2H, d, J = 7.8 Hz), 6.98 (H14a, 1H, t), 6.94 (H2 and H7, 2H, t, J = 7.4 Hz),

3.42 (H3a, 2H, t, J = 8.7 Hz), 3.02 (H5a, 3H, s), 2.95 (H2a, 2H, t, J = 8.1 Hz).

13C-{H} NMR (DMF-d7): δ 159.6, 149.8, 138.9, 136.6, 129.6, 129.5, 128.8, 121.4,

120.7, 117.3, 114.7, 74.0, 46.6, 42.5, 31.1.

Anal. (C21H19N7·0.25 THF) C, H, N: calcd, 67.50, 5.40, 25.04; found, 67.55, 5.14,

25.40.

1-(Acridin-9-yl)-3-methylimidazolidin-2-imine Hydrobromide, 43. To a

solution of compound 36 (251 mg, 1.0 mmol) in 10 mL of dry CH2Cl2 was added 0.2 mL

of triethylamine. After the yellow solution was cooled to - 10 oC, BrCN (112 mg, 1.05

mmol) was added slowly to the stirred solution. The temperature was not allowed to rise

above - 5 oC. When the addition was complete, the mixture was stirred for another 1.5 h.

Solvent was removed, and the residues was washed with a small amount of ice-cold

water and dried in vacuum. After recrystallization from ethanol, compound 43 was

obtained as a yellow microcrystalline solid. Yield: 221 mg (62%); mp 270 – 271.5 oC.

1H NMR (D2O): δ 8.48 (4H, overlapping d), 8.40 (2H, t), 8.09 (2H, t), 4.46 (H7a,

2H, m), 4.32 (H6a, 2H, m), 3.29 (H5a, 3H, s).

13C-{H} NMR (D2O): δ 157.4, 148.1, 141.4, 138.6, 130.4, 125.3, 124.2, 120.8,

51.3, 49.8, 32.4.

ESI-MS (MeOH, +ve mode) m/z: 277.0 [M-H]+.

Anal (C17H17N4Br·0.1C2H5OH) C, H, N: calcd, 57.10, 4.90, 15.48; found, 56.73,

4.73, 15.28.

46

N-Methyl-N′-tert-butoxycarbonylthiourea, 45 and tert-butyl-tert-

butoxycarbonylcarbamothioyl(methyl)carbamate, 46. A mixture of N-methyl-thiourea

(44, 1.69 g, 18.7 mmol) and NaH (0.9 g, 22.5 mmol, 60% in mineral oil) in 550 mL of

dry THF was stirred for 15 min under Ar at 0 oC. To the solution was added di-tert-butyl

dicarbonate (4.25 g, 19.5 mmol), and stirring was continued for another 30 min. A white

slurry formed within 30 min, which was stirred for another 3 h at room temperature. The

reaction mixture was quenched with 50 mL of saturated aqueous NaHCO3, poured into

450 mL of water, and extracted with ethyl acetate (5 × 150 mL). The combined organic

layers were dried over Na2SO4 and concentrated in a vacuum. The reaction mixture was

separated on an alumina column using ethyl acetate/hexane (1:5) to afford 2.11g (59%) of

45 (Rf = 0.39), and 0.95 g (17%) of 46 (Rf = 0.50) as white and pale yellow solids,

respectively.

Characterization of 45:

1H NMR (CDCl3): δ 9.68 (1H, br s), 7.92 (1H, br s), 3.17 (3H, d), 1.48 (9H, s).

13C-{H} NMR (CDCl3): δ 180.5, 151.9, 83.7, 32.0, 28.0.

Characterization of 46:

1H NMR (CDCl3): δ 12.14 (1H, br s), 3.58 (3H, s), 1.55 (9H, s), 1.51 (9H, s).

13C-{H} NMR (CDCl3): δ 181.2, 154.4, 150.1, 85.4, 82.4, 31.3, 28.0, 27.9.

tert-Butyl-{[2-(acridin-9-ylamino)ethyl]methylamino(methylamino)-

methanimine}carbamate, 47. Boc-protected thiourea 45 (460 mg, 2.41 mmol),

precursor 36 (668 mg, 2.66 mmol), and 0.75 mL of triethylamine were dissolved in 12

mL of dry N, N-dimethylformamide (DMF). The solution was cooled to 0 oC and solid

47

HgCl2 (723 mg, 2.66 mmol) was added. The mixture was stirred for 1 h at this

temperature and another 4 h at room temperature until the yellow slurry had turned black.

DMF was removed at 30 oC in vacuum, and the residue was redissolved in ethyl acetate,

filtered through Celite and dried over Na2SO4 overnight. After the solvent was removed,

the crude product thus obtained was purified by flash chromatography on an alumina

column using methanol/ethyl acetate/hexanes (1:2:2), which yielded 47 as a yellow

powder. Yield: 730 mg (74%).

1H NMR (MeOH-d4): δ 8.27 (2H, br), 7.51 (4H, br), 7.19 (2H, br), 4.02 (2H, t),

3.70 (2H, t), 2.80 (3H, s), 2.65 (3H, s), 1.37 (9H, s).

13C-{H} NMR (MeOH-d4): δ 161.1, 132.3, 78.9, 38.0, 30.5, 29.3, 26.0, 24.3.

1-[2-(Acridine-9-ylamino)ethyl]-1,3-dimethylguanidine Dihydrochloride, 38.

Compound 47 (730 mg, 1.79 mmol) was dissolved in 200 mL of 2 M HCl, and the

mixture was stirred at room temperature for 12 h. Acid was removed under vacuum, and

the resulting residue was dissolved in a minimum amount of ethanol. Ether was added

into the solution to precipitate the product as a yellow microcrystalline solid, which was

filtered and dried in a vacuum at 60 oC for 2 days. Yield: 510 mg (73%); mp 260.5–261.5

oC.

1H NMR (D2O): δ 8.11 (H1 and H8, 2H, d, J = 8.6 Hz), 7.95 (H3 and H6, 2H, t, J

= 6.9 Hz), 7.61 (H4 and H5, 2H, d, J = 8.6 Hz), 7.55 (H2 and H7, 2H, t, J = 6.9 Hz), 4.32

(H2a, 2H, t, J = 5.6 Hz), 3.59 (H3a, 2H, t, J = 5.4 Hz), 2.37 (H5a, 3H, s), 2.12 (H9a, 3H,

s).

48

13C-{H} NMR (D2O): δ 159.3, 156.4, 139.2, 136.1, 124.9, 124.5, 118.9, 112.4,

50.6, 46.2, 36.1, 27.9.

ESI-MS (MeOH, +ve mode) m/z: 308.1[M-H]+.

Anal. (C18H23Cl2N5·H2O·0.5 C2H5OH) C, H, N: calcd, 54.15, 6.69, 16.62; found,

53.69, 6.46, 16.48.

2.2.3 Crystallization and Data Collection

The X-ray intensity data were measured on a Bruker SMART APEX CCD area

detector system using MoKα radiation. The structure was solved and refined using the

Bruker SHELXTL software package (version 6.12). Crystallographic data and selected

bond distances and angles for 38, 40, 42 and 43 are summarized in Appendix B.

2.2.4 Biological Characterization: pKa Determinations, DNA Binding Affinities

and Cell Proliferation Assay

pKa measurements

The pKa values of 38, 40 and 43 were determined spectrophotometrically using a

Hewlett Packard 8354 spectrophotometer equipped with a Peltier temperature control and

a cell stirring module. Titrations were carried out at 25 oC in the pH range 2–13 with a

micro combination electrode placed inside the cuvette. Solutions of the acridine

derivatives (~50 μM) in 10-1 M HCl/100 mM NaCl were titrated with aliquots of 0.4 M

KOH, and UV-Vis spectra were recorded 2 min after each addition of base. The

protonation state of the chromophores was deduced from spectral changes around

isosbestic points. pKa’s were determined graphically from plots of pH vs.

49

log[k(Aacr/AacrH+)] (k = εacrH

+/εacr) using suitable absorbance maxima. Reported pKa’s are

means of three measurements.

Competitive ethidium displacement (fluorescent intercalator displacement assay)

The fluorometric assays have been described before [120]. The concentration of

calf thymus DNA (CT-DNA) was determined by UV-vis spectrophotometry at room

temperature, after annealling at 85 oC, to ensure accurate concentration determination.

Each well of a 96-well assay plate was loaded with aqueous buffer (6 mM Na2HPO4, 2

mM NaH2PO4, 0.1 mM Na2EDTA and 500 mM NaCl, pH 7.0) containing 37.8 μM

ethidium bromide and 15 μM(bp) CT-DNA.

The C50 values are defined as the drug concentrations which reduced the

fluorescence of the DNA-bound ethidium by 50% and are reported as the mean from

duplicate determinations. Fluorescence intensities were determined on BioRad UV CCD

Camera. Apparent equilibrium binding constants were calculated from the C50 values

using Kapp = (1.26/C50) × Ke, where Ke = 107 M-1 for ethidium bromide at pH 7 [120] .

Cytotoxicity Assay

The cytotoxicity studies were carried out using the Celltiter 96 Aqueous Non-

Radioactive Cell Proliferation Assay kit. HL-60 leukemia cells and H460 lung cancer

cells were kept in a humidified atmosphere of 5% CO2 at 37 oC. Cells were plated on 96-

well plates with 700 cells/well for H460 and 27500 cells/well for HL-60 cells. Stock

solutions of 38, 40 and 43 were prepared in phosphate-buffered saline (PBS). The cells

growing in log phase were incubated with appropriate serial dilutions of the drugs in

50

duplicate for 72 h. To each well were added 20 μL of MTS/PMS solution, and the

mixtures were allowed to equilibrate for 3 h. The absorbance at 490 nm was recorded

using a plate recorder (none of the compounds tested absorbs at this wavelength). The

reported IC50 data were calculated from non-linear curve fits using a sigmoidal dose-

response equation in GraphPad Prism and are averages of two individual experiments.

2.3 Results and Discussion

2.3.1 Two Unusual Reactivity Features of the 9-Aminoacridine Chromophore

When undertaking a survey of the guanidine synthetic organic literature, it

appeared that the target molecule might be accessible via direct transformation of the

thiourea sulfur in ACRAMTU into guanidine imine. The thiourea derivative, 37, can be

conveniently synthesized by reacting N-acridin-9-yl-N′-methylethane-1,2-diamine (36)

with methylisothiocyanate (Figure 27) [100]. Several guanidylation reactions have been

described that involve Lewis acid-promoted desulfuration [121], oxidation [122, 123], or

alkylation [124] of thiourea sulfur and subsequent reaction of the reactive intermediate

with ammonia or a primary/secondary amine. Unfortunately, all attempts to generate the

target molecule directly from compound 37 using the above-mentioned methodologies

were unsuccessful.

The first attempt to prepare target molecule 38 involves desulfuration of 37 with

phosgene, followed by ammonolysis of the resulting chloroformamidinium salt at low

temperature (Figure 27) [125]:

51

Figure 27. First attempt to generate ACRAMGU (38) by desulfurating 37 with phosgene which leads to unexpected cyclized compound 40.

As anticipated, the highly nucleophilic thiourea sulfur in 37 reacts with phosgene

to produce a (very hygroscopic) Vilsmeier-type salt. However, instead of the desired

subsequent reaction with ammonia, the reaction leads to cyclization due to the

intramolecular nucleophilic attack of the exocyclic 9-amino nitrogen on the reactive

formamidinium carbon, giving compound 40 (Figure 27), which was isolated as the

guanidinium chloride salt. The second equivalent of HCl released in this condensation is

bound by the added base, NH3.

Next, the stepwise build-up of the guanidine moiety starting from precursor 36

was attempted. The strategy involved sequential replacement of imidazole in

di(imidazole-1-yl)methanimine (41), a versatile guanidylating reagent [119], by (i) the

secondary amino group in 36 and (ii) methylamine (Figure 28). Di(imidazole-1-

yl)methanimine (41) was obtained by treating cyanogen bromide with imidazole

according to a modified literature procedure [126].

N

N

N

NH ClN

NHN

HNS

COCl2/THF/NH3/0 oC

1 23456

7 8

4a

6a7a

8a

76%

N

NHN

HNCl Cl

NH3/MeCN

0 oCN

NHN

HNH2N Cl

NH3 /MeCN

0 oC

NH4Cl

76%

N

NHHN

MeNCS/EtOH/Δ

37

40

38

36

52

Figure 28. Second attempt to generate ACRAMGU (38) by sequential replacement of imidazole in 41 by 36 and methylamine, which leads to unexpected spiro compound 42.

According to the literature [119], sequential reaction of 41 with the appropriate

amines was expected to lead to the desired, unsymmetrically substituted guanidine, 38.

Reactions of 41 with one equivalent of secondary alkylamine at room temperature were

shown to afford the monosubstituted intermediates in 60–80% yield, which could be

isolated and reacted with a second equivalent of amine to give the desired guanidine

[119]. The by-product generated is water-soluble imidazole, which can be easily removed

by repeated washings with water and saturated NH4Cl. However, in the case of amine 36,

this reaction leads to the formation of an unusual spirocyclic product, 42 (Figure 28),

which indicated that two equivalents of reagent 41 were consumed during the synthesis

of 42. Retrosynthetic analysis of compound 42 suggests that the first equivalent most

likely yielded a cyclized product, 41′, similar to compound 40, which could not be

isolated because the species reacts readily with a second equivalent of 41 via release of

one imidazole group to afford 42. Initially, equimolar amounts of 36 and 41 were reacted,

N

NHHN

N

N

NHN

NH

N

N

N

NN

N

N N NN

NH

2a3a

5a11a13a

14a

1 23456

7 8

47%

THF, r.t.

N N NN

NH

N

NHN

NH

NNNH2M

e

N

NHN

NH

HN

41

41′ 42

36 38

53

and the stoichiometry was subsequently optimized by applying two equivalents of

reagent 41.

Another strategy involved conversion of the dangling secondary amino group in

36 into cyanamide using cyanogen bromide [127] at -10 oC in the presence of a base. The

goal was to treat the reactive intermediate with methylamine to produce guanidine 38.

However, as in the previous cases, the secondary amino-group (N9) attached to the

acridine chromophore acted as an internal nucleophile, resulting in cyclization leading to

compound 43 (Figure 29), the hydrobromide salt of the nonisolable intermediate 41′

implicated in the formation of 42 (Figure 28).

Figure 29. Third attempt to generate ACRAMGU (38) using cyanogen bromide, followed by addition of methylamine, which leads to cyclized compound 43.

The only strategy to avoid cyclization and feasible pathway to the target

guanidine 38 started with the treatment of N-methylthiourea (44) with (Boc)2O in the

presence of NaH (deprotonating 44) to obtain the Boc-activated N-methylthiourea (45)

[128]. Following a published procedure [129], 45 was desulfurated in the presence of

HgCl2. In this reaction, the Boc group plays an important role as an electron-withdrawing

group rather than that of a protecting group. It activates the carbodiimide intermediate

BrCN/CH2Cl2NEt3/-10 oC

N

NHHN

N

N

NNH2 Br

1 23456

7 8

5a6a

7a62%

N

NHN

N

NH 2Me, -10 o C

one-potN

NHN

HN

NH

36

43

38

54

(Me-N=C=N-Boc) for nucleophilic addition of the dangling secondary amino group in

acridine-amine 36. The Boc-modified intermediate 47 can be easily deprotected in dilute

acid, giving the dihydrochloride salt of 1-[2-(acridin-9-ylamino)ethyl]-1,3-

dimethylguanidine (38) (Figure 30).

Figure 30. Synthetic scheme for ACRAMGU, 38.

During the various attempts of making ACRAMGU (38), two unusual reactivity

features of the 9-aminoacridine chromophore have been revealed, which can be

considered the result of the high affinity of exocyclic N9 for the activated formamidinium,

guanidyl, and cyanamide carbon centers, resulting in the formation of compounds 40, 42,

and 43, respectively. Cyclization giving a five-membered ring is apparently favored over

reaction with external nucleophiles (amines), prohibiting the formation of compound 38.

Furthermore, the inherently basic conditions in these systems produced by the external

amines (40, 43) or by the basic guanidyl imino group (42) itself have the potential to

deprotonate the 9-amino nitrogen, thereby increasing the nucleophilicity of N9. While

formation of five-membered cyclic guanidines from 1,2-diamines has been observed

N

NHHN

NH

NHN

HNH2N

H2N NH

S1. NaH/THF/0oC2. (Boc)2O/THF N

HNH

S

O

O

Et3N/HgCl2/DMF/0oCN

NHN

HNNO

O2M HClr.t.

2 Cl

(major, 59%)

NH

N

S

O

O

O

O

(minor, 17%)

1 23456

7 8

2a3a

5a

9a

73%

+

44 45 46

36 47 38·2HCl

55

previously [130], cyclization of 36 should be unfavorable because of the loss of

resonance stabilization between the N9 and the aromatic system, as evidenced in the

molecular structures of 40 and 43 (see Figure 32 and related discussion). The disruption

of N9 conjugation has profound consequences for the basicity, DNA affinity, and

biological activity of these agents.

On the other hand, transformation of C9 in 41′, the free-base form of 43

implicated in the formation of 42, into an sp3-hybridized spiro carbon, demonstrates that

this ring atom is highly susceptible to nucleophilic attack.

Figure 31. Proposed mechanism of formation of 42.

Figure 31 shows the proposed mechanism of formation of 42. The first step

involves nucleophilic attack of the guanidine nitrogen in 41′ on reagent 41 and release of

imidazole (Im). This produces an intermediate in which the modified 9-guanidyl residue

and the acridine chromophore are essentially orthogonal to each other, positioning the

imino nitrogen in close proximity to the reactive C9. In the final step, (concerted)

proximal nucleophilic attack of the imino nitrogen on C9 and proton transfer to N10 then

leads to the formation of a six-membered dihydrotriazine ring to afford the spiro

compound, 42. Spiro compounds incorporating the (9-amino)dihydroacridine moiety are

42

NH

N

N

N

NN

N

N

N

NHN

NHN

NN

N

- ImH

N

N

NNHN

N

N

9910

41′

41

56

rare, and only structures modified with five-membered heterocyclic rings at the 9-

position have been reported in the literature [131, 132].

The molecular structures of compounds 38, 40, 42 and 43 were determined by

single-crystal X-ray crystallography (Appendix B). In the structure of 40 (Figure 32),

which was first predicted on the basis of scalar connectivities in heteronuclear 2-D NMR

spectra, the acridine and guanidinium rings adopt an almost perpendicular orientation

with an angle of 97.88o between mean planes. The 9-amino nitrogen deviated

significantly from planarity, as can be expected for a partial sp2→sp3 rehybridization due

to disruption of conjugation with the polyaromatic ring system. Characteristically,

nitrogen is displaced by 0.244 Å from the plane through the adjacent carbon atoms. It is

noteworthy to mention that the exocyclic guanidine nitrogen but not the 9-aminoacridine

is protonated in this molecule. Similar structural details are observed in the guanidinium

cation of compound 43 (Figure 32).

Figure 32. Crystal structures of 40 (chloride salt, left) and 43 (bromide salt, right).

57

The crystal structure of compound 42 confirms the spirocyclic nature of this

molecule (Figure 33). It indicates the loss of aromaticity of the central ring and the

associated bent geometry of the 9,10-dihydroacridine, in which the angle between the

mean planes through the outermost phenyl rings is 26.93o. The endocyclic N1 atom is

protonated and can be considered amine-like rather than pyridine-like. Finally, the

structure of dicationic 38 in the solid state confirms the desired open-chain structure

containing a protonated classical 9-aminoacridine moiety and a terminal guanidinium

group (Figure 33).

Figure 33. Crystal structures of 42 (left) and protonated 38 (right, anions not shown).

2.3.2 Biological Evaluation: pKa Determinations, DNA Binding Constants, and

Cytotoxicities

Compounds 38, 40, and 43 were further characterized in solution to assess their

utility as DNA targeted agents, and their cytotoxicities were determined in H460 lung

58

cancer cells. Unlike the acridinium/guanidium salts, spiro compound 42 proved to be

insoluble in biologically relevant buffers, consistent with the fact that none of its nitrogen

atoms is protonable under physiological conditions, and it was not included in this study.

Relevant data are summarized in Table 2.

Table 2. Summary of Chemical and Biological Characterization for 38, 40 and 43. Compound pKa1, pKa2

a Kappb (M-1) IC50

c (μM) 38 8.9, >12 1.5 × 104 10.26 40 2.6, 9.4 < 103 >100 43 2.7, 7.9 5.1 × 103 >100

a Spectrophotometric and 1H NMR spectroscopic pH titrations. pKa1 and pKa2 reflect the protonation of the endocyclic nitrogen and guanidine nitrogen, respectively. b Apparent binding constant in calf thymus DNA. c Concentrations needed to inhibit proliferation of H460 lung cancer cells by 50%; average of two experiments.

pKa1 = 8.9, pKa2 > 12 pKa1 = 2.6, pKa2 = 9.4

Figure 34. pH titrations for compounds 38 and 40 monitored by UV-visible spectroscopy (pH range 2–12).

[40]2+ [40]+ [40]pH = 2 pH = 6 pH = 11

[38]2+ [38]+

pH = 2 pH = 11

300 350 400 0

0.1

0.2

0.3

0.4

0.5

0.6

450 500

Wavelength (nm)

300 350

400

450

0

0.05

0.1

0.15

450

0

0.05

0.1

0.15

0.2

0.25

0.3

500

Wavelength (nm)

Wavelength (nm) Wavelength (nm)

Abs

orba

nce

Abs

orba

nce

59

The pKa values of the guanidinium analogues were determined using UV-visible

spectroscopy. Changes in absorbance were monitored as a function of pH, and pKa values

were extracted using a graphical method based on the Henderson–Hasselbalch equation.

A plot of pH-dependent UV-visible traces for compounds 38 and 40 is shown in Figure

34.

An important finding in this study is that compounds 40 and 43 exist as

monocations at physiological pH, whereas derivative 38 forms a dication. This can be

attributed to the dramatically decreased proton affinities of the acridine chromophores’

endocylic nitrogen in 40 and 43 compared to 38 (Table 2), a consequence of the

disruption of conjugation between the acridine chromophores and the 9-amino nitrogens,

which have become part of the cyclic guanidinium moieties. Therefore, compound 38 is

the only derivative containing an acridine chromophore that is protonated at

physiological pH, and it is the most promising candidate for generating a hybrid agent

with DNA binding properties similar to PT-ACRAMTU (pKa, ACRAMTU = 9.8) [109].

These critical differences in basicity and protonation state ultimately affect the

DNA binding and biological activity of the molecules. Negatively charged DNA interacts

strongly with highly positively charged drugs under physiological conditions, which

often leads to the most effective inhibition of DNA–enzyme interactions and potent

biological activity. This is in agreement with the observation that the classical 9-

aminoacridine derivative 38 shows the highest DNA-binding affinity and proves to be the

only compound showing an appreciable cytotoxic effect in H460 lung cancer cell in vitro.

In addition to the less favorable electrostatics in 40 and 43, the steric hindrance produced

60

by the guanidyl groups may not allow efficient intercalation of the acridine moiety into

the DNA base stack, potentially limiting the agents’ cytotoxic potential.

2.3.3 Attempted Conjugation of ACRAMGU (38) with Monofunctional Platinum

Although a nitrogen analogue of ACRAMTU (37) could be synthesized, namely

by changing its thiourea group to a guanidine group in ACRAMGU (38), several attempts

of generating the corresponding platinum conjugate were unsuccessful. Reaction of the

free-base form of ACRAMGU, generated from the hydrochloride salt under basic

conditions, with silver-activated platinum precursor yields a complex in which both

chloro leaving groups have been replaced with a chelating (bidentate) guanidinato ligand.

This undesired binding mode, which has been observed previously in similar complexes

formed by N-H-acidic thioureas [116, 117], should render platinum unreactive with DNA

nitrogen, because it lacks a chloro leaving group. The unexpected reactivity of

ACRAMGU (38) was established by 195Pt-NMR spectroscopy: the chemical shift of –

2570 ppm found for the reaction product is typically observed for Pt2+ coordinated by 4

nitrogen donors. A value 100–150 ppm further downfield would have been expected for

a [N3Cl] environment of platinum [133].

Figure 35. Reactivity of ACRAMGU (38) with activated [Pt(en)Cl2].

NH

NHN

HNH2NCl

Cl

PtNH2H2N

Cl DMF

+1.Base

(NO3) / DMFN

HN

N

HN

N

PtNH2

H2N

195Pt NMR(D2O): δ -2570

2.

+

ACRAMGU·2HCl (38·2HCl)

61

2.4 Conclusions

In conclusion, in a successful effort to synthesize the guanidine analogue of the

anticancer active 9-aminoacridine derivative ACRAMTU, unexpected reactivity features

of the acridine C9–N9 linkage were encountered. On the basis of our observations, new

methodologies were developed that allow the formation of two novel structural motifs in

this class of compounds: (i) incorporation of N9 into five-membered cyclic guanidinium

group and (ii) transformation of C9 into a spiro carbon as part of a triazine-type

heterocycle. Further application of these methodologies may lead to novel acridines of

potential biological interest. While interesting chemistry was discovered, the ultimate

goal of making PT-ACRAMGU was not reached. Alternative linker groups are therefore

needed to overcome the inherent chelating properties of the guanidinato group. In

Chapter 3, it will be demonstrated that this can be achieved by introducing an amidine

functionality in place of the guanidine group.

62

CHAPTER 3

SYNTHESIS, CHARACTERIZATION, AND BIOLOGICAL EVALUATION OF

AMIDINE DERIVATIVES OF PT-ACRAMTU

The work contained in this chapter was initially published in Journal of Medicinal

Chemistry in 2008 and 2009. The manuscripts, including figures and schemes, were

drafted by Zhidong Ma and Dr. U. Bierbach and edited by Dr. U. Bierbach before

submission to the journal. Since the publications, changes in both format and content

have been made to maintain a consistent format throughout this work. The experiments

described were performed by Zhidong Ma, except for that the crystal structure

determinations which were contributed by Dr. C. S. Day and the in vivo evaluation,

which was performed by Washington Biotechnology Inc.

63

3.1 Background

In Chapter 2, attempts of generating the corresponding platinum conjugate of the

guanidine-modified acridine, ACRAMGU (38), failed due to the chelating properties of

the guanidinato group. Therefore, our attention was focused on other imine-based

ligands (X = NH in Figure 36), such as isoureas, isothioureas and amidines, which lack

a reactive, deprotonable NH group. In these groups, while the overall structure of

ACRAMTU is conserved, one nitrogen atom of thiourea (Y) is replaced with oxygen,

sulfur, or carbon, respectively (Figure 36). Reaction of N-(acridine-9-yl)-N´-

methylethane-1,2-diamine (36) with cyanate [134] and silicon isothiocyanate [135] and

subsequent selective methylation [136] of urea-oxygen and thiourea-sulfur yields

isourea and isothiourea derivatives, respectively (unpublished data). However, after

successful preparation of these two ligands, conjugation reactions with platinum failed,

which was attributed to either chelation or competitive binding between nitrogen and

sulfur to platinum in the case of the isothiourea.

X = S, Y = NH (thiourea derivative, "ACRAMTU") X = NH, Y = O (isourea derivative) X = NH, Y = S (isothiourea derivative) X = NH, Y = CH2 (amidine derivative)

Figure 36. Alternative strategies for generating platinum–imino analogues of PT-ACRAMTU (31).

HN

HN N Y

XPt

64

The goal of designing a compound containing a suitably modified acridine

derivative whose geometry would closely mimic that of ACRAMTU was accomplished

by introducing an amidine group (Figure 36; X = NH, Y = CH2). After undertaking a

survey of the amidine synthetic organic literature, it appeared that the target molecule

should be accessible via addition of acridine–amine 36 to propionitrile. Nucleophilic

additions to nitriles are greatly facilitated by protonating the cyano nitrogen or by binding

the nitrile to transition metal ions (Lewis acids), such as Cu(I) [137] or Sm(II) [138], to

increase the electrophilicity of the adjacent carbon atom. Unfortunately, all attempts to

generate the desired ligand using the above-mentioned methods were unsuccessful.

The synthesis of the target platinum conjugate succeeded when platinum-bound

propionitrile was reacted with precursor 36, which allows the carrier ligand to be

synthesized and attached to the platinum moiety, [PtCl(en)]+, in one step [139-141].

Details of the new synthetic approach, which resulted in a platinum–acridine conjugate

exhibiting dramatically improved chemical stability and DNA affinity, and, most

importantly, superior biological activity compared to PT-ACRAMU in vitro and in vivo,

are reported in this chapter.

3.2 Experimental Section

3.2.1 Materials and Chemicals




operating 75.5 MHz. Chemical shifts (δ) are given in parts per million (ppm) relative to

65


sodium salt (DSS) for samples in D2O. 1H and 13C NMR spectra for the new compounds

(52, 53, 54 and 55) are available in Appendix A. 195Pt NMR spectra were recorded on a

Bruker DRX-500 MHz spectrometer at 107.5 MHz. Aqueous K2[PtCl4] was used as

external standard, and 195Pt chemical shifts are reported vs [PtCl6]2-. The target

compounds (54 and 55) were fully characterized by gradient COSY and 1H-detected

gradient HMQC and HMBC spectra recorded on a Bruker DRX-500 MHz spectrometer.

Elemental analyses were performed by Quantitative Technologies Inc., Madison, NJ. All

reagents were used as obtained from commercial sources without further purification

unless indicated otherwise. Solvents were dried and distilled prior to use.

3.2.2 Synthesis and Characterization

Cis-Pt(EtCN)2Cl2, 48. Propionitrile, C2H5CN (1 mL, 14 mmol), was added into a

solution of 1.0 g of K2PtCl4 (2.4 mmol) in 15 mL of water. The solution was heated to 70

–80 oC for 4 h. The resulting yellow solid was filtered off, washed, and dried under

vacuum. The dried crude product was then purified on a silica column using

CHCl3/(CH3)2CO (1:1) as eluent, to yield cis-isomer 48 (0.5 g, 63%).

1H NMR (CDCl3) δ 2.86 (4H, q, J = 7.6 Hz), 1.41 (6H, t, J = 7.5 Hz).

Pt(EtCN)(C19H22N4)Cl2, 50. A suspension of 48 (375 mg, 1 mmol) in 20 mL of

CH2Cl2 was treated with 36 (251 mg, 1 mmol) at -10 oC. After 6 h the solution was

allowed to warm up to room temperature and stirred for another 4 h. The reaction mixture

was concentrated to a small volume and then treated with cold ether. A yellow precipitate

was collected and separated on a preparative alumina plate using hexanes/CH2Cl2 (2:3) as

66

eluent. The band of interest was collected and extracted with CH2Cl2. Slow evaporation

of the solvent gave 50 as single crystals suitable for X-ray crystallography. 1H NMR

spectrum taken of this fraction shows an unexpected doubling of signal sets, indicating

the presence of two geometrical isomers (cis and trans) in solution. From this mixture,

the trans-isomer crystallized. No further attempts to separate this mixture were

undertaken.

[PtCl(en)(EtCN)]Cl, 52. The complex [PtCl2(en)] 51 (200 mg, 0.613 mmol) was

heated at reflux in dilute HCl (pH 4) with propionitrile (2.7 mL, excess) until the yellow

suspension turned into a colorless solution (~2 h). Solvent was removed by rotary

evaporation, and the pale yellow residue was dissolved in 7 mL of dry methanol. The

solution was passed through a syringe filter, and the colorless filtrate was added directly

into 140 mL of vigorously stirred dry diethyl ether, affording 52 as an off-white

microcrystalline precipitate, which was filtered off and dried in a vacuum. Yield: 210 mg

(90%).

1H NMR (D2O) δ 2.88 (2H, q, J = 7.5 Hz), 2.64 (4H, m), 1.30 (3H, t, J = 7.5 Hz).

13C-{H} NMR (D2O) δ 122.9, 48.7, 48.4, 12.3, 9.2.

195Pt NMR (D2O) δ −2711.

Anal. (C5H13Cl2N3Pt) % C, H, N: calcd 15.76, 3.44, 11.02; found 15.73, 3.06,

10.74.

[PtCl(NH3)2(EtCN)]Cl, 53. This precursor was synthesized analogously to 52

starting from [PtCl2(NH3)2] (300 mg, 1 mmol) and propionitrile (4.2 mL). Yield: 295 mg

(83%).

67

1H NMR (D2O) δ 2.89 (2H, q, J = 7.5 Hz), 1.31 (3H, t, J = 7.5 Hz).

13C-{H} NMR (D2O) δ 121.9, 12.3, 9.2.

195Pt NMR (D2O) δ -2467.

Anal. (C3H11Cl2N3Pt) % C, H, N: calcd 10.15, 3.12, 11.83; found 10.09, 2.90,

11.69.

[PtCl(en)(C19H23N4)](NO3)2. 54. Precursor complex 52 (170 mg, 0.45 mmol)

was converted to its nitrate salt by reaction with AgNO3 (75 mg, 0.44 mmol) in 10 mL of

anhydrous DMF. AgCl was filtered off, and the filtrate was cooled to −10 °C. N-

(Acridin-9-yl)-N´-methylethane-1,2-diamine (36) [100] (117.4 mg, 0.47 mmol) was

added to the solution, and the suspension was stirred until it turned into an orange-red

solution (~7 h). The reaction mixture was added dropwise into 200 mL of cold

dichloromethane, and the resulting yellow slurry was vigorously stirred for 30 min. The

precipitate was recovered by membrane filtration, dried in a vacuum overnight, and

dissolved in 40 mL of methanol containing 1 mol equiv of HNO3. After removal of the

solvent by rotary evaporation, the crude product was recrystallized from hot ethanol,

affording 54 as a microcrystalline solid. Yield: 169 mg (52%).

1H NMR (DMF-d7) δ 13.92 (1H, s), 9.90 (1H, s), 8.70 (2H, d, J = 8.6 Hz), 8.07

(4H, overl m), 7.63 (2H, t, J = 6.8 Hz), 5.78 (2H, s), 5.48 (2H, s), 4.51 2H, t, J = 6.3 Hz),

4.10 (2H, t, J = 6.7 Hz), 3.21 (3H, s), 3.12 (2H, q, J = 7.4 Hz), 2.68 (4H, s), 1.33 (3H, t, J

= 7.5 Hz).

13C-{H} NMR (DMF-d7) δ 170.4, 159.0, 140.6, 135.7, 128.2, 124.3, 119.4, 113.5,

50.1, 49.4, 49.2, 47.5, 28.0, 11.4.

195Pt NMR (DMF-d7) δ −2494.

68

UV/Vis (H2O): λmax 413, ε = 10571.

Anal. (C21H33ClN8O6Pt·H2O) % C, H, N: calcd 34.08, 4.49, 15.14; found 34.16,

4.31, 15.15.

[PtCl(NH3)2(C19H23N4)](NO3)2, 55. This analogue was prepared as described for

52 starting from 293 mg (0.83 mmol) of 53, 132 mg (0.79 mmol) of AgNO3, and 197 mg

(0.79 mmol) of N-(acridin-9-yl)-N´-methylethane-1,2-diamine. Yield: 315 mg (57%).


(4H, overl m), 7.62 (t, J = 7.2 Hz), 6.27 (1H, s), 4.53 (3H, s), 4.49 (2H, t, J = 6.8 Hz),

4.16 (3H, s), 4.10 (2H, t, J = 6.3 Hz), 3.20 (3H, s), 3.15 (2H, q, J = 7.6 Hz), 1.33 (3H, t, J

= 7.5 Hz).

13C-{H} NMR (DMF-d7) δ 170.3, 159.3, 140.8, 135.9, 126.5, 124.5, 119.6, 113.6,

50.8, 47.8, 28.3, 11.5.

195Pt NMR (DMF-d7) δ −2264.

UV/Vis (H2O): λmax 413, ε = 9224.

Anal. (C19H29ClN8O6Pt·2.5H2O) % C, H, N: calcd 30.79, 4.62, 15.12; found 30.66,

4.14, 15.03.

Complexes 52´ and 54´ containing 15N-en were synthesized accordingly starting

from [PtCl2(15N-en)] (51´).

[PtCl(15N-en)(EtCN)]Cl, 52´.

1H NMR (MeOH-d4): δ 6.11 and 5.86 (2H, d of t, NH2 trans to Cl, 1J(1H–15N) =

75 Hz, 3J(1H–1H) = 5.3 Hz), 6.01 and 5.76 (2H, d of t, NH2 trans to N, 1J(1H–15N) = 75

Hz, 3J(1H–1H) = 5.2 Hz), 2.93 (2H, q, J = 7.6 Hz), 2.57 (4H, m), 1.33 (3H, t, J = 7.5 Hz).

69

[PtCl(15N-en)(C19H23N4)](NO3)2, 54´.


(4H, m, overlap), 7.63 (2H, t, J = 6.8 Hz), 6.26 (NH, 1H, s), 5.82 and 5.53 (2H, d of t,

NH2 trans to Cl, 1J(1H–15N) = 74.5 Hz, 3J(1H–1H) = 5.0 Hz and 5.1 Hz), 5.47 (2H, d of t,

NH2 trans to N, 1J(1H–15N) = 74.7 Hz, 3J(1H–1H) = 5.1 Hz), 4.51 (2H, t, J = 6.3 Hz),

4.10 (2H, t, J = 6.7 Hz), 3.21 (3H, s), 3.12 (2H, q, J = 7.4 Hz), 2.68 (4H, s), 1.33 (3H, t, J

= 7.5 Hz).

3.2.3 Cell Proliferation Assay

The cytotoxicity studies were carried out according to a standard protocol [142]

using the Celltiter 96 Aqueous Non-Radioactive Cell Proliferation Assay kit. HL-60

leukemia cells and H460 lung cancer cells were kept in a humidified atmosphere of 5%

CO2 at 37 oC. Cells were plated on 96-well plates with a total 700 cells/well for H460 and

27500 cells/well for HL-60 cells.

Stock solutions of PT-ACRAMTU (31), 54 and 55 were prepared in phosphate-

buffered saline (PBS) and serially diluted with media prior to incubation with cancer cells.

The cells growing in log phase were incubated with appropriate serial dilutions of the

drugs in duplicate for 72 h. To each well was added 20 μL of MTS/PMS solution, and the

mixtures were allowed to equilibrate for 3h. The absorbance at 490 nm was recorded

using a plate recorder (none of the derivatives tested absorbs at this wavelength). The


response equation in GraphPad Prism (GraphPad Software, La Jolla, CA) and are

averages of four individual experiments.

70

3.2.4 Crystallization and Data Collection

The X-ray intensity data were collected on a Bruker SMART APEX CCD area

detector system using MoKα radiation. The structures were solved and refined using the

Bruker SHELXTL software package (version 6.12). Crystallographic data and selected

bond distances and angles for 50 and 54 are summarized in Appendix B.

3.2.5 NMR Spectroscopy

Time-dependent NMR spectroscopy. NMR spectra in arrayed experiments

were collected at 37 °C on a Bruker 500 DRX spectrometer equipped with a triple-

resonance broadband inverse probe and a variable temperature unit. Reactions were

performed in 5-mm NMR tubes containing 2 mM platinum complex 54 and 6 mM 5´-

guanosine monophosphate (5´-GMP) or 2 mM complex 54 and 2 mM N-acetylcysteine

(N-AcCys) (10 mM phosphate buffer, D2O, pH* 6.8). The 1-D 1H kinetics experiments

were carried out as a standard Bruker arrayed 2-D experiment using a variable-delay list.

Incremented 1-D spectra were processed exactly the same, and suitable signals were

integrated. Data were processed with XWINNMR 3.6 (Bruker, Ettlingen, Germany).

The concentrations of platinum complex at each time point were deduced from relative

peak intensities, averaged over multiple signals to account for differences in proton

relaxation, and the data were fitted to the equation y = A0 × e−x/t (where A0 = 1 and t−1 =

kobs) using Origin 7 (OriginLab, Northampton, MA).

2-D NMR spectroscopy. 2-D 1H-15N NMR experiments were performed with

15N-(en)-labeled complex, 54´, by setting up an alternating array of 1-D 1H

WATERGATE (water suppression by gradient-tailored excitation) and 1H-15N HMQC

71

experiments. Experiments were performed in 5 mm NMR tubes at 37 °C in 10 mM

sodium phosphate buffer made in 90% H2O/10% D2O (pH 6.8). The 1-D 1H

WATERGATE pulse sequence was modified to incorporate a delay (d31). Each delay

was set to collect data at 20-min intervals, followed by acquisition of a 2-D 1H-15N

HMQC data set. Each HMQC were collected with 2 scans per time increment. A sweep

width of 10,139 Hz in F1 and 5000 Hz in F2 was used in each experiment. All 2-D

spectra were processed to 1024 in F2 and 1024 in F1 using forward linear predication.

15N chemical shifts were referenced to an external 15NH4Cl standard (25 mM in 0.1 M

DCl). All data sets were processed with Felix 2000 (Accelrys, San Diego, CA).

3.2.6 Liquid Chromatographic Separation–Mass Spectrometry Detection

Incubations. Reactions of 54 or PT-ACRAMTU (31) with N-acetylcysteine were

performed at 1:1 drug-to-amino acid ratio in 10 mM sodium phosphate buffer (pH 7.1).

Incubations were performed at 37 oC for 6 h.

Chromatographic separations. The mixtures were separated by reverse-phase

HPLC using the LC module of an Agilent Technologies 1100 LC/MSD Trap system

equipped with a multi-wavelength diode-array detector and autosampler. A 4.6 × 150 mm

reverse phase Agilent ZORBAX SB-C18 (5 µm) analytical column was used in all the

assays, which was maintained at 25 oC during separations. One wavelength, 413 nm, was

used to monitor acridine containing fragments. The following eluents were used for the

separations: solvent A, degassed water/0.1% formic acid, and solvent B, methanol/0.1%

formic acid. The gradient for separation was solvent A changing from 98% to 30% over

72

30 min at a flow rate of 0.5 mL/min. The integration of peaks was done using the

LC/MSD Trap Control 4.0 data analysis software.

Mass spectrometry. Mass spectra were recorded on an Agilent 1100LC/MSD ion

trap mass spectrometer. After separation by in-line HPLC, adducts were infused into the

atmospheric-pressure electrospray source. Ion evaporation was assisted by a flow of N2

drying gas (350 oC) at a pressure of 40 psi and a flow rate of 10 L/min. Positive-ion mass

spectra were recorded with a capillary voltage of 2800V and a mass-to-charge scan range

of 150 to 2200 m/z.

3.2.7 Confocal Fluorescence Microscopy

Cell line. The human ovarian carcinoma cell line A2780 was obtained from

Sigma Aldrich and was used for all fluorescence microscopy studies. Cells were

maintained in exponential growth phases as monolayers in Advanced DMEM media

supplemented with 2% fetal calf serum, 1% Antibiotic Antimyotic and 1% glutamine at

37 oC in 5% CO2. PBS and trypsin were obtained from Invitrogen.

Instrumentation and materials. Confocal images were aquired using a Nikon

DE-Eclipse microscope C1 equipped with Coherent Radius 405-25, Sapphire 488.20 and

Melles Griot 561 optically pumped semiconductor laser systems. Fluorescence imaging

was performed using an Olympus CellR System, equipped with an MT20 illumination

system. The excitation filters used were 403/12, 492/18, 572/23 nm (DAPI/FITC/Texas

RED) and emission filters 435/475, 510/550 and 595/700 nm (DAPI/FITC/Texas RED).

The objective used was an UPLAN Super Apochromat 40X, NA 0.9 with correction

collar (0.11-0.23). LysoTracker Red DND-99 lysosome marker and MitoTracker Green

73

FM were obtained from Invitrogen. Cells were treated with hydrogen peroxide to induce

apoptosis.

Incubations. A2780 cells were seeded in 6-well plates containing glass coverslips.

Approximately 2 × 105 cells were seeded in each well with 2 mL of media, before being

incubated at 37 oC in 5% CO2 for 24 h. For colocalization studies, the cells were then

dosed with either PT-ACRAMTU (31) or 54 at a concentration of 20 mM, and incubated

for another 10 min. LysoTracker Red DND-99 (1 μM) or MitoTracker Green FM (1 μM)

was added to drug-containing cells and untreated cells. For apoptosis studies, cells were

treated with either, 1) drug (20 mM), 2) hydrogen peroxide (6.6 μL, 30%), or 3) drug (20

mM) plus hydrogen peroxide (6.6 μL, 30%), and then incubated for 10 min. Coverslips

were mounted on glass slides and sealed with nail polish, and images were collected

within 10 min of slide preparation.

3.2.8 H460 Xenograft Study

H460 xenografts were established in nude athymic female mice via bilateral

subcutaneous injections. Treatment began when the average tumor volume was

approximately 100 mm3. The tumor-bearing mice were randomized depending on tumor

volume into three groups of five test animals each: one control group receiving vehicle

only, one group treated at 0.1 mg/kg 5d/w (A), and one group treated at 0.5 mg/kg q4d

(B). Animal weights and tumor volumes were measured and recorded for 17 days after

the first dose was administered. Tumor volumes were determined using the formula: V

(mm3) = d2 × D/2, where d and D are the shortest and longest dimension of the tumor,

respectively, and are reported as the sum of both tumors for each test animal. At the end

74

of the study, all animals were euthanized and disposed off according to Standard

Operating Procedures (SOPs). One tumor per test animal as well as lungs and kidneys

were removed during necropsy and shipped to the Bierbach lab for detection of platinum

levels in each tissue. The xenograft study was performed by Washington Biotechnology

Inc. (Simpsonville, MD), PHS-approved Animal Welfare Assurance, # A4912-01.

Statistical analysis of the growth curves was done using a non-linear polynomial random-

coefficient model in SAS Proc Mixed (SAS Institute Inc., Cary, NC).

3.2.9 Inductively Coupled Plasma–Mass Spectrometry Detection of Platinum

Drugs and Standards. Nitric acid (70%, trace-metal grade) and hydrochloric

acid (35% trace-metal grade) standards were obtained from Fisher. Platinum standard, as

well as internal indium and bismuth were obtained from High-Purity Standards. All

standards and spike solutions were prepared and stored in PFA containers.

Aqueous calibration standards were prepared by diluting 10 μg/mL platinum

single-element ICP–MS stock solutions (High-Purity Standards with 2% HCl).

Quantification of element concentration was performed using external calibration with

aqueous standard solutions and internal standardization using 115In and 209Bi (at 10 ppb in

the final solution) as internal standards. The Pt isotopes used for calculation of Pt

concentrations were 194Pt and 195Pt.

Sample preparation. Tissue samples (tumor, kidneys, and lungs) were thawed

and used for determination of total Pt in tissues. The samples were blotted to remove

extra fluid, weighed, and placed in a solution containing 2.5 mL of 70% nitric acid and

2.5 mL of 35% hydrochloric acid. The samples were digested at 60 oC for 16 h. The

75

aqueous digestate was diluted to 25 mL using distilled deionized water. After the samples

were centrifuged for three minutes at 3000 rpm, an aliquot of 4 mL of supernatant was

mixed with 0.04 mL internal standard containing 1 ppm indium and bismuth. The

solution was transferred to 15 mL tubes for measurement of Pt by inductively coupled

plasma mass spectrometry on a Thermo ICP–MS instrument at the Analytic Chemistry

Service facility of the Research Triangle Institute, Research Triangle Park, NC.


3.3.1 Lewis Acid Assisted Addition Reaction

Prior to the successful synthesis of the amidine analogue of PT-ACRAMTU (31),

a test reaction was performed to determine the feasibility and conditions of the platinum-

mediated amidination. The susceptibility of platinum-bound nitriles to nucleophilic attack

by water, amines, alcohols and thiols yielding amidates, amidines, iminoethers and

iminothioethers has been reported in the literature [143-146]. Based on the reported

reactivity of acetonitrile complexes of cisplatin [146], a reaction scheme leading to the

desired ligand, N-[2-acridin-9-ylamino]ethyl]-N-methylpropionamidine, can be proposed

(Figure 37). The strategy involves addition of the secondary dangling amino group in N-

(acridine-9-yl)-N´-methylethane-1,2-diamine (36) to the propionitrile ligand in complex

48.

76

Figure 37. Test reaction for addition of secondary amine to platinum-bound nitrile.

To generate cis-bis(propionitrile)dichloroplatinum(II) (48), propionitrile was

added in large excess to an aqueous solution of K2PtCl4, and the reaction mixture was

stirred for several hours at 70-80 oC. The product, which was contaminated with the

trans-isomer, was purified by column chromatography. One nitrile ligand in complex 48

is then converted into amidine by reaction with N-(acridine-9-yl)-N´-methylethane-1,2-

diamine (36) at low temperature to produce a mixture of cis- and trans-[PtCl2(EtCN)(N-

[2-acridine-9-ylamino]ethyl)-N-methylpropionamidine] (49 and 50), indicating cis–trans

isomerization. While the geometric isomers could not be separated chromatographically,

the trans-isomer, 50, crystallized selectively from the mixture and produced crystals

suitable for X-ray crystallography. The crystals structure of complex 50 confirms the

formation of desired amidine coordinative linkage (Figure 38). An interesting feature in

this structure proves to be the protonation state of the acridine moiety. The proton of the

exocyclic 9-amino group (N3) has migrated to endocyclic N4, producing the unusual

N

HNNH

K2PtCl4H2O/C2H5CN

55oCPt

Cl Cl

N NCH2Cl2 / -10oC

PtCl Cl

NC

NH NNH

N PtCl

Cl NH NNH

NN

C

+

48

49 50

77

imino form of the acridine, which is stabilized by an intramolecular hydrogen bond

formed between N1 and N3.

The formation of the platinum–amidine bond in complex 50, which confirmed the

feasibility of the proposed addition chemistry, encouraged us to pursue the same strategy

for the synthesis of the amidine analogue of PT-ACRAMTU from the appropriate

monofunctional platinum–nitrile precursors.

Figure 38. Molecular structure of trans-[PtCl2(EtCN)(N-[2-acridin-9-ylamino]ethyl]-N-methylpropionamidine)] 50 with selected atoms labeled.

3.3.2 Design Rationale

Using the methodology developed in the previous section, the desired acridine, N-

[2-acridin-9-ylamino]ethyl]-N-methylpropionamidine, was synthesized and introduced as

78

a ligand in one step in place of ACRAMTU (Figure 39). In analogy to the synthesis of

complex 50, the reaction involved addition of the secondary amino group in N-(acridin-9-

yl)-N´-methylethane-1,2-diamine (36) across the activated CN bond [147] of platinum-

bound propionitrile (EtCN). The amidination reactions were performed with the

appropriate platinum precursors (52, 53) derived from complexes [PtCl2L2] (L2 = en, 51;

L = NH3, 14) to afford the corresponding platinum–acridine hybrids, which were isolated

in their fully protonated forms as the water soluble dinitrate salts, 54 and 55. NH3, instead

of en, was introduced as a nonleaving group to increase the solubility of the conjugate.

Figure 39. Synthesis of target compounds. Reagents and conditions: (i) EtCN/H2O, 70 oC, 2 h. (ii) (1) AgNO3, DMF, rt; (2) N-(acridine-9-yl)-N´-methylethane-1,2-diamine (36), DMF, -10 oC, 7 h; (3) HNO3, MeOH

The structure of 54, as determined by X-ray crystallography (Appendix B),

confirms the formation of the desired amidine coordinative linkage and overall complex

geometry, which shows platinum in a square planar environment defined by a single

chloride leaving group, a bidentate en nonleaving group, and amidine nitrogen (Figure

40). A comparison of the molecular structures of 54 and PT-ACRAMTU reveals a critical

difference between the amidine and thiourea coordination modes: while the bond angle at

PtL L

Cl Cl+ C2H5CN Pt

L L

Cl N Cl

NNH

HN

NH

NH

NHNPt

L L

Cl

2+

+

(NO3)285% 50-60%

14/51 52/53 54/55

51, 52 and 54: L2 = en; 14, 53 and 55: L = NH3

i ii

79

the donor atom (N1) in 54 is 129.6(4)o, the corresponding angle at sulfur in PT-

ACRAMTU is considerably more acute (103.8(4)o) [102]. The opening of this bond angle

in 54 relieves steric hindrance in the coordination sphere of the metal, which has

previously been identified as a rate-limiting factor in reactions of PT-ACRAMTU with

DNA nitrogen [102, 111].

Figure 40. Molecular structure of 54 with selected atoms labeled. Nitrate counterions are not shown. Thermal ellipsoids are drawn at the 50% probability level.

3.3.3 Biological Activity

The newly synthesized conjugates 54 and 55 were studied, along with the

prototype, PT-ACRAMTU (31), for their cytotoxic effects in the human leukemia cell

line, HL-60, and in the NSCLC cell line, NCI-H460. The results of the cell proliferation

assay are summarized in Table 3. In HL-60 cells, complex 54 showed moderate activity

80

similar to PT-ACRAMTU, based on an IC50 value in the micromolar range. Complex 55,

in which the en group was replaced with the solubility-enhancing ammine ligands, was

approximately 6-fold more potent in this cell line than 54. All three compounds

performed significantly better in H460 cells. This cell-line specific enhancement is most

pronounced for compound 54, which proves to be 100-fold more active in the solid tumor

cell line than in the leukemia cell line. Most strikingly, complexes 54 and 55 were an

order magnitude more cytotoxic in H460 cells than PT-ACRAMTU. This is significant

because PT-ACRAMTU has previously demonstrated only slightly better activity in this

cell line than cisplatin (IC50 of 0.63 μM in clonogenic survival assays [103]).

Table 3. Cytotoxicity Data (IC50

a, μM) for Platinum–Acridine Conjugates. Compound HL-60 (μM) H460 (μM) PT-ACRAMTU(31) 3.95 ± 0.24 0.35 ± 0.017 54 2.97 ± 0.11 0.028 ± 0.0024 55 0.47 ± 0.064 0.026 ± 0.0022

aConcentrations of compound that reduce cell viability by 50% determined by cell proliferation assays. Cells were incubated with platinum for 72 h. Values are means of four experiments ± the standard errors of the mean. Complexes 54 and 55 are remarkably cytotoxic in H460 NSCLC cells. The only

platinum-based agent known to inhibit H460 cell growth with similar potency in the

nanomolar concentration range is [(trans-PtCl(NH3)3(trans-Pt(NH3)2

(NH2(CH2)6NH2)2))](NO3)4 (BBR3464, 24) (discussed in Chapter 1, Figure 18), a

trinuclear platinum complex currently in clinic trials against various cancers. Cisplatin is

at least 20-fold less potent than the nonclassical agents in H460 cells, with IC50 values

typically in the micromolar range [103].

81

3.3.4 Kinetics of Platinum Binding with Biologically Relevant Nucleophiles

Kinetics of DNA Platination. To test the effect of the modification made to the

prototypical agent on the metal´s activity with DNA, the reactions of PT-ACRAMTU (31)

or 54 with excess 5´-guanosine monophosphate (5´-GMP) were monitored by 1H NMR

spectroscopy in a simple model system mimicking DNA nitrogen. Compound 54 reacts

significantly faster with 5´-GMP than PT-ACRAMTU (Figure 41).

PT-ACRAMTU 31: X = S, Y = NH 54 : X = NH, Y = CH2

Figure 41. Kinetic study using time-dependent 1H NMR spectra for the reaction of PT-ACRAMTU (31) and complex 54 with 5´-GMP monitored for aromatic guanine and acridine protons giving signal assignments. R´ stands for ribose and phosphate. (A) Reaction scheme with important atoms labeled.

A

PtH2N NH2

Cl X

Y

NNH

NH

2+

1234

5678

910

N

N

O

NH

NH2NR'

8+

O

OH OH

OPO

ONaNaO

5'-GMP

PtH2N NH2

X

Y

NNH

NH123 4

5678

910

N

N

O

HN

H2N NR'

Cl

8

R' =

82

Figure 41 (Continued). (B) Stacked plot for the reaction monitored for PT-ACRAMTU (31). (C) Stacked plot for the reaction monitored for 54.

B C

H1/H8 H3/H6 H4/H5 H2/H7G-H8

H1/H8 H3/H6 H4/H5 H2/H7G-H8

H1/H8 H3/H6 H4/H5 H2/H7G-H8

H1/H8 H3/H6 H4/H5 H2/H7G-H8(br) Time(h)

---0---

---2---

---8---

---17---

Reactant(↓) Reactant (↓)

Adduct(↑) Adduct (↑)

8.6 8.5 8.4 8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 ppm 8.6 8.5 8.4 8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 ppm

83

From pseudo first-order treatment of the kinetic data, rate constants (kobs, 37 oC)

of 1.77 × 10-4 s-1 and 4.93 × 10-5 s-1 were calculated, which correspond to half-lives of 65

and 234 min for 54 and PT-ACRAMTU, respectively (Figure 42). Detailed analysis of

the 1H NMR spectra indicates that compound 54 forms monoadducts, in which guanine-

N7 has selectively substituted the chloride ligand, whereas the platinum–amidine linkage

proves to be resistant to nucleophilic attack by nucleobase nitrogen in the presence of

excess nucleotide, in complete analogy to PT-ACRAMTU [110, 111].

Figure 42. Progress of the reaction of PT-ACRAMTU (31) (circles) and 54 (triangles) with the mononucleotide 5´-GMP at 37 oC monitored by 1H NMR spectroscopy. The inset shows a scheme of the reaction monitored. The data plotted is the mean of two experiments. Rate constants and half lives have been calculated for PT-ACRAMTU and 54, respectively.

65 min 1.77 × 10-4 s-154

234 min4.93 × 10-5 s-1PT-ACRAMTU (31)

Half Life (t1/2)Rate constant (kobs)

84

A 2-D [1H, 15N] heteronuclear multiple quantum coherence (HMQC) NMR study

performed with 15N-labeled complex (54´) supports this mechanism and suggests that

adduct formation is most likely preceded by aquation of the Pt–Cl bond, which is

commonly observed in platinum drug–DNA interactions [98]. HMQC NMR spectra were

recorded at each time point for 54´ to elucidate the substitution chemistry at the platinum

center and to detect possible hydrolysis products. 15N-labeled en as a non-leaving group

is a sensitive probe to detect structural changes in the coordination sphere of platinum:

15N chemical shifts of N-donor ligands attached to platinum (and other metals) are

sensitive to changes in the coordination sphere, especially substitution of ligands trans to

the observed 15N nucleus.

The [1H,15N] HMQC spectrum recorded of 54´ in phosphate-buffered H2O/D2O

(90%/10%) shows two cross-peaks at δ 4.98/-34.59 ppm and 5.19/-34.61 ppm. These

have been assigned to the NH2 group trans to the amidine nitrogen and to the NH2 group

trans to the chloro leaving group, respectively, on the basis of the greater 15N deshielding

effect of chloride compared to the nitrogen (Figure 43, left). In the 2-D spectrum

recorded after 4 h of incubation at 37 oC, two new cross-peaks for the putative hydrolysis

product are observed (Figure 43, right). Only a small amount of complex 54´ turned into

hydrolyzed product, in agreement with a reversible reaction that lies far to the right. The

cross-peaks at δ 5.01/-51.91 ppm and 5.38/-34.32 ppm in this spectrum can be assigned

to the NH2 group trans to water oxygen (least deshielding) and to the NH2 group trans to

amidine nitrogen, respectively.

85

Figure 43. Hydrolysis of complex 54´ detected by 2-D [1H-15N] HMQC NMR spectroscopy giving cross-peak assignments.

The 2-D [1H, 15N] HMQC study was designed to detect the potential hydrolysis

intermediate in reaction mixtures containing 54´ and excess 5´-GMP in phosphate buffer

(Figure 44). However, detection of such a species was unsuccessful, which might be

attributable to the low abundance of the hydrolyzed intermediate due to rapid substitution

of this weak leaving group by 5´-GMP (see Figure 41). Complex 54´ and its

monofunctional guanine adduct were the only species detected in the time-dependent

HMQC spectra (Figure 44). In the adduct, the NH2 groups trans to G-N7 and trans to

amidine NH experience similar trans-influences from the sp2 nitrogens, resulting in

overlapping cross-peaks.

NH

NH

NHNPt

H2N NH2

Cl

2+

NH

NH

NHNPt

H2N NH2

H2O

3+

AB

H2O

86

t = 100 min t = 180 min

artifact

A&BA&B

-35.66/5.33 and -35.93/5.37 NH2 trans to G-N7 and amidine NH

artifact

t = 0 min t = 40 min

artifact artifact

-34.59/4.98 NH2 trans to N

-34.61/5.19 NH2 trans to Cl

A&B

NH

NH

NHNPt

H2N NH2

Cl

2+

NH

NH

NHNPt

H2N NH2

N7

3+

AB5'-GMP

GMP

Figure 44. Reaction of complex 54´ with 5´-GMP monitored by 2-D [1H-15N] HMQC NMR spectroscopy giving cross-peak assignments.

87

The simple modification made to PT-ACRAMTU (31) resulted in greatly

enhanced DNA binding kinetics. While the exact mechanism for the amidine analogue 54

and the associated rate laws are yet to be determined, amidine nitrogen appears to

accelerate associative substitution reactions, most likely due to steric factors favoring

nucleophilic attack (either from water or DNA bases) on the metal. Hydrogen bonding

between the imino hydrogen of the complex and exocyclic groups of the DNA bases,

such as guanine-O6 [98], may also contribute to the rate enhancement. Efficient DNA

binding of the new derivatives is most likely a major contributor to the greatly improved

biological activity in rapidly proliferating H460 cells.

Reactivity with cysteine sulfur. In complete analogy to the nucleotide binding

study discussed in the previous section, the reaction of the new conjugate (54) with

cysteine sulfur was tested. In arrayed one-dimensional 1H NMR experiments, the reaction

of 54 with one equivalent of N-acetylcysteine (N-AcCys) (25 oC, 10 mM phosphate

buffer, D2O, pH* 6.8) was found to proceed considerably more slowly than the reaction

of PT-ACRAMTU under the same conditions. The relative rates at which the amino acid

reacted with 54 and PT-ACRAMTU were deduced from the change in integral intensities

of 1H NMR signals assigned to the platinum complexes (Figure 45).

88

PT-ACRAMTU 31: X = S, Y = NH

54 : X = NH, Y = CH2 Figure 45. Time-dependent 1H NMR spectra for the reaction of PT-ACRAMTU (31) and complex 54 with N-AcCys. Drug-based proton signals integrated for plotting the concentration–time traces are highlighted with black boxes. (A) Reaction scheme with protons monitored highlighted.

PtH2N NH2

Cl X

Y

NNH

NH

2+

+HNHS

OHO

O

N-acetylcysteine

en

linkage N-acetylcysteine

Products (see Fig. 48)

A

89

Figure 45 (Continued). (B) Stacked plot for the reaction monitored for PT-ACRAMTU (31). (C) Stacked plot for the reaction monitored for 54.

4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 ppm 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 ppm

CH2CH2 (linkage) CH2CH2 (en) CH2 (linkage) CH2CH2 (en) B C

0 h

2 h

8 h

18 h

Time

90

Under the conditions chosen, the reaction between 54 and N-AcCys follows a

second-order rate law, a typical mechanism that is dominated by direct substitution of

chloride ligand by cysteine sulfur and is not limited by the rate of aquation [148]. In

contrast, the more rapid reaction of PT-ACRAMTU with N-AcCys followed neither

second-order nor first-order kinetics, suggesting that the reaction of this derivative

proceeds via a more complicated mechanism (Figure 46). In both cases, the 1H NMR

spectra of the mixture indicated formation of multiple products. Attempts to assign the

species formed by 54 and PT-ACRAMTU using 2-D [1H-15N] NMR spectroscopy, a

technique applied in nucleotide binding studies of [15N]-en labeled conjugates, 31 and 54

(see Section 3.3.4), were unsuccessful due to the complexity of the reaction mixtures

[111].

Figure 46. Progress of the reactions of 54 (blue circle) and PT-ACRAMTU (red square) with one equivalent of N-AcCys at 25 oC monitored by 1H NMR spectroscopy. The data shown is the mean of two experiments. The solid line (for 54) represents a curve fit assuming second-order conditions, [Pt]/[Pt]0 = (1 + k[Pt]0t)-1; (t1/2 = 180 min). The dashed line (for PT-ACRAMTU, 31) has no physical significance.

0 200 400 600 800 10000.0

0.2

0.4

0.6

0.8

1.0

[Pt]/

[Pt] 0

Time(min)

91

The reactivity of Pt–Sthiourea bond and its cis-labilizing effect on chloride are most

likely the cause of the increased cysteine reactivity of PT-ACRAMTU compared to

analogue 54. The rationale for incorporating a thiourea-based nonleaving group in the

original design was to enhance the reactivity of the metal with DNA nucleobase by

exploiting the cis-activating effect of sulfur. In their pioneering studies of the inorganic

kinetic cis-effect, Tobe and coworkers demonstrated for the complexes [PtCl(en)(dmso-

S)]+ and [PtCl(dms)]+ (dmso = dimethylsulfoxide, dms = dimethylsulfide) that the sulfur

donors greatly accelerate chloride substitution by an incoming nucleophile relative to

analogous complexes with [N3Cl] donor sets [116, 117]. It was also demonstrated that the

relative enhancement (kS/kN) is large for strong incoming nucleophiles, such as sulfur and

some nitrogen ligands, but negligible for weak ligands like water. The comparative

kinetic study of amidine-modified complex 54 and thiourea-based PT-ACRAMTU 31

shows that replacement of sulfur with an imino donor group reduces, as expected, the

metal´s reactivity with sulfur, but enhances its binding with DNA nitrogen. These

findings strongly suggest that, unlike the reaction with cysteine, the reaction with DNA is

not controlled by the cis-effect but most likely by steric hindrance and/or hydrogen

bonding between the imino hydrogen of the complex and exocyclic groups of the DNA

base that favor complex aquation and facilitate nucleophilic attack of nucleobase nitrogen

on the metal center (see discussion of 5´-GMP binding in section 3.3.4).

Thus, efficient DNA binding of the new derivatives (54 and 55) and reduced

levels of detoxification compared to PT-ACRAMTU are most likely the major

contributors to the greatly improved biological activity in rapidly proliferating H460 cells.

However, subtle structural differences at the target level that may render specific lesions

92

induced by 54 and 55 more cytotoxic than those induced by PT-ACRAMTU cannot be

ruled out.

3.3.5 Analysis of Platinum–Amino Acid Products

In the kinetic study of reactions of these two drugs with N-AcCys, the 1H NMR

spectra of the mixtures indicated formation of multiple products. But attempts to assign

the species formed by PT-ACRAMTU (31) and 54 using two dimensional [1H–15N]

NMR spectroscopy were unsuccessful due to the complexity of the reaction mixtures. To

overcome this difficulty, LC-MS was used to analyze the product distribution in mixtures

of platinum complexes incubated with one equivalent of sulfur nucleophile (37 oC, 6 h,

10 mM phosphate buffer, pH 7.2). The two most abundant products formed by 54 were

successfully separated (Figure 47A) and unambiguously identified as the dinuclear

complex [{Pt(en)(L)}2(μ-N-AcCys*)]4+ ([P2]4+, 72%) (Figure 47B) and mononuclear

species [Pt(en)(L)(N-AcCys*)] ([P3]+, 14%) (Figure 47C) in electrospray mass spectra

recorded in positive-ion mode. (The asterisk indicates the dianionic form of metal-bound

N-AcCys at neutral pH. L = N-(2-(acridine-9-ylamino)ethyl)-N-methylpropionamidine,

acridinium cation).

These findings suggest that adduct P3, formed by substitution of chloride by

cysteine sulfur, rapidly reacts with the unreacted complex 54 to form dinuclear species,

P2 (Figure 49). The tendency of cysteine sulfur to readily induce bridged complexes with

platinum(II) drugs has been amply demonstrated [148]. The platinum–amidine linkage

and the en chelate in complex 54 appears to be resistant to nucleophilic attack by cysteine

93

based on the presence of a minor amount of free acridine L (P1, 3%) and the absence of

ring-opened adducts.

Figure 47. (A) Reverse-phase HPLC elution profile monitored at 413 nm of a mixture of complex 54 incubated with N-AcCys. The asterisks indicate an acridine-containing species that could not be assigned on the basis of mass spectral data.

0 5 10 15 20 25 Time [min] 0

5

10

15

mAU

03060803.D: UV Chromatogram, 405-421 nm

**, 11%

P1, 3%

P2, 72%

P3, 14%

Inte

nsity

A

94

Figure 47 (Continued). Positive-ion electrospray mass spectra of adducts P2 (B) and P3 (C) showing peaks for the molecular ion, [P3]+ (m/z 725.2) and [P2-2H]2+ (m/z 643.7, z = 2) and fragments resulting from in-source collisionally activated dissociation (CAD). The insets illustrate the observed fragmentation patterns. The discrepancy between the calculated and observed m/z values for some of the platinum containing ions is due to distorted Pt isotope patterns.

252.1

307.2

490.6

562.2

643.7674.0

726.2

0.00

0.25

0.50

0.75

1.00

1.25

106

200 300 400 500 600 700 800 900 m/z

Inte

nsity

630 640 660 670 680

[P2]4+ = 1286.4

HN

NH

N NHPt

H2N NH2

S

NH

NH

NHNPt

NH2H2N

HNCHC

H2C

OO

O

726.2

490.6

674.0

z = 2 z = 1

221.0252.1

307.2

363.1

418.0

725.2

0

1

2

3

4

5

6

107

200 300 400 500 600 700 800 900 m/z

HN

NH

N NHPt

H2N NH2

S

HN CH C

CH2OH

OO

418307

221.0

252.1

[P3] + = 723.8

Inte

nsity

P2 P3 B C

95

In contrast, the LC-MS data acquired for PT-ACRAMTU 31 reveal a more

complicated reaction pattern (Figure 48A–C). In analogy to complex 51, the chloride

substitution pathway leads to dinuclear adduct, [{Pt(en)(ACRAMTU)}2(μ-N-AcCys*)]4+

(P1´, 32%) (Figure 48B), most likely via the intermediate [Pt(en)(ACRAMTU)(μ-N-

AcCys*)]+, which could not be detected in this case. In addition, significant amounts of

ACRAMTU (P3´, 33%) and the bisintercalator complex [Pt(en)(ACRAMTU)2]4+ (P2´,

26%) (Figure 48C), were observed in the HPLC profiles (Figure 48A). Formation of the

latter complex, P2´, can be explained with chloride substitution in PT-ACRAMTU by

free ACRAMTU in the reaction mixture. The release of ACRAMTU by N-AcCys was

unexpected and shows that thiourea, a typical nonleaving group in reactions of PT-

ACRAMTU with DNA nitrogen [107, 110], becomes substitution-labile in the presence

of cysteine sulfur. These reactions have been summarized in Figure 49.

96

Figure 48. (A) Reverse-phase HPLC elution profile monitored at 413 nm of a mixture of PT-ACRAMTU 31 incubated with N-AcCys. The asterisks indicate an acridine-containing species that could not be assigned on the basis of mass spectral data.

0 5 10 15 20 25 30 35 40 45 Time [min]

0

2

4

6

8

10

mAU

UV Chromatogram, 405-421 nm

P1’, 32%

P2’, 26%

P3’, 33%

**

A

97

Figure 48 (Continued). Positive-ion electrospray mass spectra of adducts P1´ (B) and P2´ (C) showing peaks for the molecular ion, [P1´-2H]2+ (m/z 660.2, z = 2) and [P2´]+ (m/z 903.4) and fragments resulting from in-source collisionally activated dissociation (CAD). The insets illustrate the observed fragmentation patterns. The discrepancy between the calculated and observed m/z values for some of the platinum containing ions is due to distorted Pt isotope patterns.

HN

NH

N S

NH

PtH2N NH2

S

NH

NH

NS

HN

PtNH2H2N

HNCHC

H2C

HOO

O

741578.3704

325

221.1 252.2

291.1

325.2

371.2 420.2

484.6 578.3 660.2

704.1

741.2

0

1

2

3

4

5

6

x106

200 300 400 500 600 700 800 900 m/z

Inte

nsity

[P1´]4+ = 1322.4

B P1’ HN

NH

N S

NH

NH

NH

NS

HN

Pt

H2N NH2

578

518

221.0

252.1

325.2

422.2

452.2

518.2

578.2

903.4

0

1

2

3

4

x106

200 300 400 500 600 700 800 900 m/z

Inte

nsity

[P2´]+ = 903.4

P2’ C

98

Figure 49. Pathways for reaction of PT-ACRAMTU (31) and 54 with N-AcCys.

HN

NH

N S

NH

PtH2N NH2

S

NH

NH

NS

HN

PtNH2H2N

HNCHC

H2C

HOO

O

HN

NH

N S

NH

PtH2N NH2

S

HN CH C

CH2OH

OO

HN

NH

N S

NH

PtH2N NH2 NH

NH

NS

HN

HN

NH

N S

NH

PtH2N NH2

Cl N-AcCys

HN

NH

N S

NH

HN

NH

N NHPt

H2N NH2

S

NH

NH

NHNPt

NH2H2N

HNCHC

H2C

HOO

O

HN

NH

N NH

N-AcCys

HN

NH

N NHPt

H2N NH2

S

HN CH C

CH2OH

OO

HN

NH

N NHPt

H2N NH2

Cl

31

37, P3´

37

P1 (minor)

54

31

P1´

P2´

P3

54

P2

99

3.3.6 Determination of the Subcellular Localization of Complex 54

Fluorescence microscopy has become a powerful tool for detecting the subcellular

localization of fluorescent molecules and can provide insight into their cellular

interactions and mechanism of action [149, 150]. It is important to investigate the

properties of pharmacologically important compounds in intact cells, because the cellular

environment is difficult to simulate. Thus, live-cell imaging has become a popular

method in cellular studies of bioactive molecules [150]. The rapid development of

organelle-specific dyes has made the technique particularly powerful, since they can be

used to perform colocalization studies [150]. In this study, experiments were performed

with both PT-ACRAMTU (31) and complex 54. While both compounds shared similar

intracellular distributions, complex 54 showed a significantly higher level of fluorescence

in all of the experiments. Therefore, in the following sections, only representative

imaging data acquired for complex 54 will be discussed.

To locate relevant subcellular organelles, such as lysosomes and mitochondria,

and to study their shapes and patterns, A2780 ovarian cancer cells were treated with

either LysoTracker Red or MitoTracker Green. LysoTracker Red is a lysosomal stain

consisting of a fluorophore attached to a weak base, which readily accumulates in acidic

cellular compartments (low internal pH), and, thus, is useful for investigating the

biosynthesis and pathogenesis of lysosomes [151]. MitoTracker, on the other hand,

contains a thiol-reactive chloromethyl moiety for labeling mitochondria. MitoTracker

Green FM passively diffuses across the plasma membrane and accumulates in active

mitochondria [152]. Another dye, Hoechst 33342, is a useful nuclear tracker because it

binds to the minor groove of DNA [153]. Unfortunately, the emission spectrum of this

100

tracker and that of 54 overlap, which prohibits the detection of colocalized dye and drug

(unpublished data).

Figure 50. Fluorescence microscopy image of A2780 cells treated with LysoTracker Red DND-99 (left) and MitoTracker Green FM (right).

Figure 50 shows the distinct differences in the appearance of the two

compartments within the cell. The lysosomes were observed to have a cytoplasmic

pattern of staining characterized by dots and points, while the mitochondria were found to

form closely packed, kidney shaped bodies.

The next goal was to confirm that complex 54 can be detected in A2780 cells, and,

if so, to determine its cellular localization. After incubating the cells with the drug for 10

min, an intense blue fluorescence is observed indicating compound 54 has entered the

cell (Figure 51). By comparison with Figure 50, it appears that the complex is localized

mainly to the lysosomes, based on the presence of large cytoplasmic punctuate areas,

which have been observed before for lysosomes that have accumulated platinum-

101

containing drugs [154-157]. However, there also seems a small amount of the drug

present in the nucleus, which indicates that free (not DNA-bound) platinum–intercalator

conjugate is present in the nucleus whose fluorescence has not been quenched by π-

stacking with DNA bases.

Figure 51. Representative fluorescence microscopy image (left) of A2780 cells incubated with complex 54 for 10 min. Enlarged image (right) of a single cell.

To prove this more conclusively and to reveal the subcelluar localization of the

complexes, colocalization studies were conducted, in which platinum-treated cells were

co-stained with LysoTracker Red and MitoTracker Green, respectively. Complex 54 was

shown in the previous experiments to be visible using the blue emission filter, while

LysoTracker Red can only be detected using the red emission filter, and MitoTracker

Green can only be observed using the green filter. Consequently, these emission filters

should allow the fluorescence originating from the platinum complex 54 and that from

MitoTracker Green /LysoTracker Red (Figure 52) to be clearly distinguished.

102

Figure 52 shows the relative distributions of complex 54 and mitochondrial

marker, MitoTracker Green. In particular, the overlay panel (III) indicates that the

distributions for this compound and mitochondria are mutually exclusive. On the other

hand, the blue-red color observable in the superposition of images obtained from co-

staining with the complex 54 and LysoTracker Red (Figure 52 C) indicates major areas of

co-localization of the drug and lysosomes.

103

Figure 52. Confocal fluorescent microscopy images of A2780 cells treated with complex 54 while co-stained with MitoTracker Green FM (top) and LysoTracker Red (bottom), respectively. (I) and (A) show images taken through blue emission filter, (II) shows an image through green emission filter, (III) shows superpositions of blue and green images, (B) shows an image through red emission filter, and (C) shows superpositions of blue and red images. The brightness and contrast of the images have been manually modified.

I II III

A B C

104

One of the possible mechanisms to account for the accumulation of the platinum

complex within the lysosomes is that the presence of protonated acridine will render the

compound susceptible to pH-dependent accumulation within the highly acidic internal

environment of lysosomes.

However, the blue fluorescence of the complexes is not restricted to the regions

showing red fluorescence, indicating that, while the complex has entered the lysosomes,

it is also present elsewhere in the cytoplasm or in other organelles. Based on the

appearance of the faint blue fluorescent areas, the platinum complex must have entered

the nucleus, which was not observed at lower concentrations (not shown). It might be that

at low drug concentrations, platinum binding to nuclear DNA is quantitative and the

drug-based fluorescence is effectively quenched [156], which renders platinum

unobservable in the nucleus. However, at higher concentrations of the platinum complex,

the DNA might become saturated with drug, resulting in a light blue fluorescence due to

the presence of the unbound conjugate. If this case, the rupturing of the nuclei and

breakdown of the chromatin structure, which releases the fluorophores, should give rise

to increased fluorescence levels. In order to prove this hypothesis, A2780 cells were

dosed with complex 54 and 1 mM hydrogen peroxide simultaneously for 10 mins, after a

period of which it is possible to identify those cells undergoing apoptosis. The results are

shown in Figure 53.

105

Figure 53. Representative fluorescence microscopy images of A2780 cells incubated with complex 54 alone (left) and co-treated with hydrogen peroxide (right) for 10 mins.

By comparing the two images in Figure 53, it appears that the cancer cells are

dying and imploding, and the areas of enhanced fluorescence correspond to the dense

circular regions of each cell, which are believed to be the nuclei. This supports the

previously described hypothesis, although it was important to verify that this enhanced

fluorescence was not a result of the distinct changes in cellular autofluorescence that are

known to accompany apoptosis [158]. Therefore, a control sample in which cells were

treated only with hydrogen peroxide for 10 mins was examined, and it showed no

autofluorescence within this time frame, unlike the sample of complex 54.

The fluorescent microscopy data confirm that our platinum–acridine conjugates

are able to enter cancer cells despite their cationic charge. The protonability of the

acridine chromophore is most likely the cause of accumulation of drug in the lysosomes.

Increased fluorescence in apoptotic cells suggests that some drug must also have entered

106

the nucleus. This supposition should be confirmed by other methods such as ICP–MS to

determine the amount of platinum associated with nuclear DNA extracted for the cells.

3.3.7 Antitumor Activity and Systemic Toxicity

The antitumor activity of 55 was evaluated against H460 bilateral tumors

implanted into athymic nude mice. Complex 55 was selected for the study because it was

slightly more soluble in biological media than 54. Complex 55 was administered

intraperitoneally (ip) according to the following dose schedule: (A) 0.1 mg/kg, five days

per week for two consecutive weeks (5d/w), and (B) 0.5mg/kg, three doses given at 4-day

intervals (q4d). The tumor volumes recorded in both treatment groups and in untreated

control animals are plotted vs days of treatment in Figure 54.

107

Figure 54. Evaluation of 55 against H460 NSCLC tumors xenografted into nude mice. Growth curves are shown for untreated control animals (open squares), and mice treated according to schedule A (filled triangles) and schedule B (filled circles). Measurement of tumor volumes began on day 0, and treatment began on day 4. Each data point represents the mean of 5 tumor volumes ± S.E.M. (A) 0.1 mg/kg, five days per week for two consecutive weeks (B) 0.5 mg/kg, three doses given at 4 days intervals.

At the end of the study, the tumors measured 1834 ± 160, 1789 ± 309 and 1102 ±

319 mm3 (mean ± SEM) for the control animals and animals treated according to

schedules A and B, respectively. On the basis of these data, the low-dose treatment A had

no effect on tumor growth. However, treatment at the higher dose B, which is close to the

maximum tolerated dose (MTD) of compound 55, slowed the tumor growth rate

significantly compared to the control group, which led to a reduction in the mean

terminal tumor volume by 40%.

The high cell kill potential of the new hybrids in H460 cells translates into

pronounced antitumor activity. This was demonstrated for compound 55 in the

0

5 15

200

500

1000

1500

2000

Control

0.1 mg/kg i.p./5d/w

0.5 mg/kg i.p./q4d

Time (days)

10

108

corresponding tumor xenograft in which the agent slowed the tumor growth at a sublethal

dose close to the MTD. The high cytotoxic potency of compound 55 is documented by

the fact that it is tolerated at doses an order of magnitude lower than those commonly

applied for cisplatin when administrated ip [159]. Because complex 55 is quite toxic at a

dose of 0.5 mg/kg, which results in significant weight loss in the treated animals, further

structural modifications are necessary to minimize adverse effects and improve the

therapeutic index of complex 55. Nevertheless, our new agent showed significantly

improved cytotoxic potential compared to the “classical” monofunctional, complex, cis-

[Pt(NH3)(pyridine)Cl]+, which requires high doses to produce appreciable antitumor

effect in vivo [160]. A recent study in kidney cell lines demonstrated that relatively

higher intracellular levels of this agent, mediated by organic cation transporters, are

required to achieve a cytotoxic effect similar to that produced by the clinical agent,

oxaliplatin [161].

3.3.8 Distribution of Platinum in Murine Tissue Samples

After completion of the antitumor activity study, biological tissues from

euthanized test animals were investigated to rationalize the high toxicity and potent

activity of 55 in the xenograft model. Specifically, accumulation levels of platinum in

various biological tissues (tumors, kidneys, and lungs) (Appendix C) at the two different

dose levels (0.1 mg/kg and 0.5 mg/kg, see 3.3.7) were determined using inductively

coupled plasma–mass spectrometry (ICP–MS) (Figure 55).

109

Figure 55. Platinum concentrations in various biological tissues (tumors, kidneys and lungs) at two different doses (0.1 mg/kg and 0.5 mg/kg) determined by ICP–MS after 16 hours of tissue digestion. There are significant differences in platinum accumulation levels between various tissues. Plotted data points are the mean of five tissue samples in five independent experiments.

The highest platinum concentration was observed in the kidneys, which was 15–

30-fold higher than in the lungs and tumors. For the low-dose treatment A (0.1 mg/kg),

the concentrations of platinum in all tissues are higher than those observed for the high-

dose treatment B (0.5 mg/kg), especially in the kidneys, which might be due to the

different treatment schedules. As described before, treatment A was performed for five

consecutive days, which would require the kidneys to excrete the toxic drug at a high rate.

However, at the higher-dose treatment, B, each injection occurred at four-day intervals,

50

2

4

6

8

10

12

14

150

200

250

LungsLungs

Kidneys

Kidneys

TumorsTumors

0.5 mg/kg0.1 mg/kg

Pt i

n Ti

ssue

s (n

g/g)

Treatment of Mice with Two Different Doses

110

which allows more time for the kidneys to excrete platinum and might lead to lower

platinum concentrations in the affected tissue.

In order to obtain a better understanding of platinum distribution, retention of

platinum in each organ was calculated as a percentage of the total administered doses

(Figure 56). Assuming an average mass of a test animal of 19 g, at the low dose treatment

A (0.1 mg/kg), a total amount of 6.92 μg of platinum was injected into each mouse, while

an average of 2.09 ng of platinum was detected in the tumors, which accounts for 0.30%

of the total injected drug. A similar amount, 1.63 ng (0.24%) was detected in the lungs.

By contrast, a dramatically larger amount of platinum, 53.47 ng (7.73%), was found in

the kidneys. Therefore, at the same dosage, the majority of platinum accumulated in the

kidneys, the major organ of excretion, which most likely contributes to the toxicity

observed in the treated group of mice.

On the other hand, at the MTD treatment B (0.5 mg/kg), the retained percentages

of platinum in these three biological tissues are all lower than in treatment A. Assuming

an average weight of 19 g per mouse, 7.98 μg of platinum was injected, while 1.42 ng

(0.18%), 1.37 ng (0.17%) and 37.57 ng (4.71%) of platinum were detected in tumors,

lungs and kidneys, respectively (Tables 4 and 5).

111

Figure 56. Retention of platinum in each organ calculated as percentage of the total administered doses (0.1 mg/kg and 0.5 mg/kg) determined by ICP–MS after 16 hours of tissue digestion. There are significant differences of platinum accumulation levels between various tissues. Plotted data points are the mean of five tissues samples in five independent experiments.

Table 4. Platinum Concentrations (ng/g) in Various Biological Tissues (Tumors, Kidneys and Lungs) at Two Different Doses (0.1 mg/kg and 0.5 mg/kg) Determined by ICP–MS after 16 Hours of Tissue Digestion. Reported Data are the Mean of Five Independent Experiments.

Dose Tumors Lungs Kidneys 0.1mg/kg 4.45 ± 0.24b,c 10.70 ± 0.47 195.46 ± 15.24 0.5mg/kga 3.33 ± 0.45 9.01 ± 1.26 136.51 ± 14.60 a Maximum Tolerable Dose. b Concentrations of platinum in tissues are in ng/g wet weight. c Mean ± Standard error of the mean (SEM).

0.0

0.1

0.2

0.3

0.4

4

5

6

7

8

9

Tumors

Kidneys

Lungs

Per

cent

age

of P

t in

Each

Tis

sue

0.5 mg/kg0.1 mg/kg

Treatment of Mice with Two Different Doses

Kidneys

Tumors

Lungs

112

Table 5. Retention of Platinum Per Single Organ Calculated as a Percentage of the Total Administered Doses (0.1 mg/kg and 0.5 mg/kg) Determined by ICP–MS after 16 Hours of Tissue Digestion. Reported Data are the Mean of Five Independent Experiments.

Dose Tumors Lungs Kidneys 0.1mg/kg 0.30 ± 0.07b,c 0.24 ± 0.02 7.73 ± 1.07 0.5mg/kga 0.18 ± 0.06 0.17 ± 0.02 4.71 ± 0.43 a Maximum Tolerable Dose. b Percentage of platinum retained in tumors, kidneys and lungs (% of total doses). c Mean ± Standard error of the mean (SEM).

On the basis of the high platinum levels established in the kidneys of mice treated

with compound 55 and the yellow discoloration of the kidneys observed during

necropsies (all other organs appeared normal, according to Washington Biotechnology,

Inc.), it seems likely that platinum toxicity to this organ may have a dose-limiting effect

and may contribute to the weight loss in the animals. However, the data also suggest that

there is no direct relationship between the levels of systemic toxicity and the absolute

amount of platinum in the kidneys: while the 0.1 mg/kg doses cause the highest platinum

levels and kidney discoloration, this treatment schedule does not lead to weight loss. On

the other hand, the kidneys removed from mice treated at the MTD of 0.5 mg/kg did not

show any sign of kidney damage. Thus, inefficient platinum excretion may not be the

major cause of the toxicity.

3.4 Conclusions and Clinical Implications

In conclusion, we have now demonstrated that a simple structural modification in PT-

ACRAMTU by replacing thiourea sulfur with imino nitrogen leads to greatly enhanced

cytotoxicity in H460 non-small cell lung cancer (NSCLC) cells and tumor growth in the

113

corresponding xenograft model. Two complexes, 54 and 55, have been synthesized using

platinum-activated amidination chemistry. Compound 55 significantly slows the tumor

growth rate in a H460 xenograft study in mice by 40% and is the first non-cross-linking

platinum agent endowed with promising activity in NSCLC in vivo.

An important issue to be addressed is the relatively high toxicity of the hybrid agent

in treated animals. The results of a necropsy performed on the test animals treated with

55 revealed “mild to yellow discoloration of the kidneys” while “everything else

appearing normal”. In the ICP–MS study, the highest platinum concentration was

observed in the kidneys. It has recently been demonstrated that the nephrotoxicity effect

of cisplatin is mediated by its GSH adducts [162]. Thus, it is possible that the GSH

adducts formed by complex 55 (see analogous cysteine adducts P2 abd P3, Figure 47 in

Section 3.7.5) contribute to the toxicity of this compound. Although compound 55

showes reduced reactivity with cysteine sulfur in comparison with PT-ACRAMTU,

additional modifications may be necessary to suppress this reactivity and improve the

compound’s metabolic stability.

It was also demonstrated that a relationship exists between the rate of DNA binding

and cytotoxic potential. A simple mononucleotide model monitored by 1H NMR was

employed to study the reactivity of PT-ACRAMTU 31 and 54 with nucleobase nitrogen.

It showed that 54 reacted considerably faster than PT-ACRAMTU did. Rapid formation

of irreversible DNA adducts can be considered an important feature in an antitumor agent

designed to kill aggressive, rapidly proliferating cancer cells.

Non-cross-linking DNA-targeted agents, such as 54 and 55, have great potential in

the treatment of cancers that do not respond to cisplatin because their adducts may evade

114

DNA repair mechanisms. A survey of the recent NSCLC literature suggests that a

correlation exists between the expression levels of nucleotide excision repair (NER)

genes in NSCLC and the progression of the disease: NSCLC patients with an excision

repair-proficient tumor phenotype respond extremely poorly to platinum drugs [163-167].

Overexpression of ERCC1, for instance, seems to be associated with tumor resistance.

The NER pathway is responsible for the repair of platinum–DNA intrastrand cross-links,

the putative cytotoxic lesions of cisplatin, which compromises the drug’s efficacy in

affected cancers. NER recognizes and removes chemically irreversible bulky lesions that

severely distort and destabilize duplex DNA [168]. Unlike cisplatin´s 1,2 intrastrand

cross-link, which severely bends DNA and reduces its thermal and thermodynamic

stability, the monofunctional intercalative binding mode established for PT-ACRAMTU

(and also expected for 54 and 55) causes local helix unwinding, but no bending, and

increases the thermal stability of the modified duplex [169, 170]. Therefore, the latter

nonclassical adducts may be poor substrates for NER and circumvent the associated

resistance mechanism in affected cancer cells, which might explain why this type of

hybrid agent performs better than cisplatin in NSCLC cells.

115

CHAPTER 4

CHANGING THE CONNECTION SITE OF THE

LINKER IN PT-ACRAMTU FROM THE 9-POSITION TO THE 4-POSITION

The work contained in this chapter was initially published in Bioorganic & Medicinal

Chemistry Letters in 2008. The manuscript, including figures and schemes, was drafted

by Zhidong Ma and Dr. U. Bierbach and edited by Dr. U. Bierbach before submission to

the journal. Since the publication, changes in both format and content have been made to

maintain a consistent format throughout this work. The experiments described were

performed by Zhidong Ma, except for the cytotoxicity assays which were contributed by

Dr. G. L. Kucera and G. Saluta.

116

4.1 Background

Chapters 2 and 3 were concerned with a structure–activity relationship (SAR) study

of PT-ACRAMTU analogues in which sulfur of thiourea attached to the 9-position of

acridine has been replaced with imino nitrogen. The approach intended to be minimally

invasive to the core structure of the conjugate, while changing its reactivity with

biologically relevant nucleophiles.

In another SAR study, we were interested in the effects of repositioning the platinum-

modified side chain in PT-ACRAMTU from the 9-position of the acridine to the 4-

position. The rationale behind this approach was that variation of the site of attachment

might have an impact on the sequence and groove specificity of DNA adducts formed by

this agent, which, in turn, might lead to altered biological activity.

Acridine derivatives carrying side chains in the 4-position of the heterocyclic base

have been studied extensively [171]. These include acridine-4-carboxamides, such as N-

[2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA), a topoisomerase-targeted

anticancer agent currently being considered as a second-line therapy for non-small-cell

lung and ovarian cancers [172].

As discussed in Chapter 1, the unique DNA damage profile produced by PT-

ACRAMTU is a consequence of the compound’s sequence preference and directionality

of intercalation, which leads to unprecedented platination of adenine bases at N3 (~10%

of adducts) in the DNA minor groove [106]. The preclinical success of PT-ACRAMTU

and the hypothesis that a relationship exists between its antitumor activity in cisplatin-

resistant cell lines and its distinct DNA damage profile have prompted several structure-

activity relationship studies.

117

Previously, modifications were made to the linker that connects the acridine and

platinum moieties [102, 103], spectator ligands on the metal center were varied [173],

and 4-substituted ACRAMTU derivatives were introduced as threading intercalators

[174]. In the present set of compounds (Figure 57), the thiourea and guanidine groups

were incorporated into a 4-carboxamide side chain. A new platinum-containing hybrid

agent was synthesized and tested along with the platinum-free carriers against HL-60 and

NCI-H460 cells.

X = S: thiourea X = N: guanidine Figure 57. Changing 9-aminoacridine into acridine-4-carboxamide derivatives. 4.2 Experimental Section

4.2.1 Materials and Methods




operating 75.5 MHz. Chemical shifts (δ) are given in parts per million (ppm) relative to


sodium salt (DSS) for samples in D2O. The platinum-free acridines 57 and 60 were fully

N

HNN N

H

XN

HNO

N NH

X9

4

49

118

characterized by gradient COSY and 1H-detected gradient HMQC and HMBC spectra

recorded on a Bruker DRX-500 MHz spectrometer. The 195Pt NMR spectrum of the

target complex 58 was recorded on a Bruker DRX-500 MHz spectrometer at 107.5 MHz.

Aqueous K2[PtCl4] was used as external standard, and 195Pt chemical shifts are reported

vs [PtCl6]2-. 1H and 13C NMR spectra for the new compounds (57, 58, 59 and 60) are

available in Appendix A. Elemental analyses were performed by Quantitative

Technologies Inc., Madison, NJ. All reagents were used as obtained from commercial

sources without further purification unless indicated otherwise. Solvents were dried and

distilled prior to use. Flash chromatography was performed using ACROS (50-200 mesh)

neutral alumina gel.

The compounds N-(2-(methylamino)ethyl)acridine-4-carboxamide (56) [175-177]

and N-methyl-N´-tert-butoxycarbonylthiourea (45) [178] were prepared according to

previously described procedures.

4.2.2 Synthesis and Characterization

N-(2-(1,3-Dimethylthioureido)ethyl)acridine-4-carboxamide, 57. A solution of

methylisothiocyanate (0.33 g, 4.55 mmol) in 10 mL of absolute ethanol was added

dropwise within 10 min to a solution of 1.0 g (3.58 mmol) of 56 in 50 mL of absolute

ethanol. The mixture was heated at reflux for 6 h and passed, while hot, through a Celite

pad. Ethanol was removed in vacuum and the residue was recrystallized from hot

methanol to afford microcrystalline 57. Yield: 0.98 g (77%); mp. 198 oC.

1H NMR (DMSO-d6): δ 11.43 (H17, t, J = 5.5 Hz), 9.33 (H9, s), 8.73 (H3, d, J =

7.1 Hz), 8.39 (H1, d, J = 8.4 Hz), 8.32 (H5, d, J = 8.8 Hz), 8.24 (H8, d, J = 8.4 Hz), 7.97

119

(H6, t, J = 6.8 Hz), 7.76 (H2, t, J = 8.1 Hz), 7.71 (H7, t, J = 8.0 Hz), 7.58 (H24, NH, b),

4.09 (H19, 2H, t, J = 6.4 Hz), 3.79 (H18, 2H, q, J = 6.1 Hz), 3.18 (H21, 3H, s), 2.91

(H25, 3H, d, J = 4.1 Hz);

13C-{H} NMR (DMSO-d6): δ 181.9, 165.2, 147.0, 145.4, 138.5, 134.5, 132.9,

131.7, 128.7, 128.4, 128.1, 126.5, 126.4, 125.6, 125.2, 51.7, 38.2, 37.2, 32.6;

Anal. (C19H20N4OS) C, H, N, S: calcd, 64.75, 5.72, 15.90, 9.10; found, 64.42,

5.63, 15.86, 9.02.

[PtCl(en)(C19H21N4OS)](NO3)2, 58. The nitrate salt of 58 was formed by adding

1.4 mL of 1 M HNO3 to 0.5 g of 57 in methanol. The solvent was removed completely in

a vacuum and the residue redissolved in 10 mL of anhydrous DMF. A mixture of 0.46 g

(1.41 mmol) of [PtCl2(en)] and 0.23 g (1.41 mmol) of AgNO3 in 10 mL of anhydrous

DMF was stirred at room temperature in the dark for 14 h. Precipitated AgCl was filtered

off through a Celite pad. The yellow solution containing 57 was combined with the

filtrate, and the mixture was stirred for 4 h in the dark. DMF was removed in a vacuum at

30 oC yielding a yellow oily residue, which was dissolved in 1.0 L of dry methanol.

Activated carbon was added, and the solution was stirred for 15 min. Carbon was filtered

off, and the solution was concentrated to a final volume of 100 mL. Crude 58 was

obtained as a bright yellow solid after the solution was stored at -10 oC for 24 h. The

crude batch was recrystallized from hot methanol. The solution was stored in the

refrigerator for 48 h to afford 58 as a microcrystalline yellow solid, which was dried at

60 oC. Yield: 650 mg (60%); mp. 185 oC.

1H NMR (MeOH-d4) : δ 9.53 (1H, s), 8.61 (1H, d, J = 7.3 Hz), 8.37 (1H, d, J =

8.5 Hz), 8.27 (1H, d, J = 8.7 Hz), 8.20 (1H, d, J = 8.5 Hz), 8.00 (1H, t, J = 8.4 Hz), 7.70

120

(2H, m), 5.28 (2H, NH2 on ethylenediamine ligand, s), 5.05 (2H, NH2 on

ethylenediamine ligand, s), 4.19 (2H, t, J = 5.9 Hz), 3.82 (2H, t, J = 6.0 Hz), 3.12 (3H, s),

2.38 (4H, C2H4 on ethylenediamine ligand, s);

13C-{H} NMR (DMF-d7): δ 176.6, 167.0, 147.8, 146.2, 140.23, 135.8, 134.1,

133.0, 129.3, 129.2, 128.3, 127.6, 127.5, 126.9, 126.0, 54.4, 50.5, 48.5, 40.2, 38.3, 34.5;

195Pt NMR (DMF-d7) δ -2859

Anal. (C21H29ClN8O7PtS · H2O) C, H, N: calcd, 32.08, 3.97, 14.25; found, 32.09,

3.86, 14.03.

ESI-MS (MeOH, +ve mode) m/z: 642.2 [M]+

tert-Butyl-((2-(acridine-4-carboxamido)ethyl)methylamino)(methylamino)-

methaniminecarbamate, 59. A mixture of 159 mg (0.92 mmol) of 45, 256 mg of 56

(0.92 mmol), and 257 μL of triethylamine was prepared in 7 mL dry DMF. The solution

was cooled to 0 oC, and 249 mg (0.92 mmol) of HgCl2 were added. The yellow slurry

was stirred for 1 h at 0 oC and for another 4 h at room temperature until the mixture

turned black. DMF was removed in a vacuum, and the residue was dissolved in ethyl

acetate, filtered through Celite, and dried over Na2SO4 overnight. After the solvent was

removed, the crude product was purified by flash chromatography on an alumina column

using methanol/ethyl acetate (2:3) as eluent. Yield: 281 mg (70%).

1H NMR(MeOH-d4) : δ 9.11 (1H, s), 8.78 (1H, d), 8.30 (2H, m), 8.14 (1H, d),

7.92 (1H, t), 7.67 (2H, m), 3.84 (2H, t), 3.75 (2H, m), 3.09 (3H, s), 2.82 (3H, s), 1.41

(9H).

((2-(Acridine-4-carboxamido)ethyl)methylamino)(methylamino)methanimi -

nium chloride, 60. To remove the Boc group, 200 mg (0.46 mmol) of 59 was dissolved

121

in 2.0 M HCl. The mixture was stirred at room temperature overnight. Acid was removed

using a rotary evaporator, and the resulting residue was dissolved in a minimum amount

of ethanol. The product was precipitated with diethyl ether and purified by flash

chromatography on an alumina column using methanol/ethyl acetate (1:1) as eluent.

Yield: 101 mg (59 %); mp: 182 oC.

1H NMR (D2O): δ 8.25 (H9, s), 8.11 (H3, d, J = 7.1 Hz), 7.71 (H6 and H8, 2H,

one triplet and one doublet overlap), 7.66 (H1, d, J = 8.1 Hz), 7.49 (H7, t, J = 7.9 Hz),

7.44 (H5, d, J = 8.4 Hz), 7.27 (H2, t, J = 7.3 Hz), 3.47 (H18, 2H, t, J = 6.0 Hz), 3.38

(H19, 2H, t, J = 6.5 Hz), 2.92 (H21, 3H, s), 2.39 (H25, 3H, s);

13C-{H} NMR (D2O): δ 168.3, 156.8, 146.6, 144.5, 138.5, 134.8, 134.3, 132.3,

128.7, 128.0, 126.8, 126.0, 125.6, 124.9, 124.7, 49.7, 36.7, 36.4, 28.2;

ESI-MS (MeOH, +ve mode) m/z: 336.4 [M]+

Anal. (C19H22ClN5O · 0.5H2O · 0.5C2H5OH) C, H, N: calcd, 59.47, 6.49, 17.33;

found, 59.04, 6.54, 16.90.

4.2.3 Biological Characterization: pKa Determinations and Cell Proliferation

Assay

pKa Measurements. The pKa value of 57 was determined spectrophotometrically using a

Hewlett Packard 8354 spectrophotometer equipped with a Peltier temperature control and

a cell stirring module. Titrations were carried out at 25 oC in the pH range 2-13 with a

micro combination electrode placed inside the cuvet. Solutions of the acridine derivatives

(~50 μM) in 50%MeOH/50% 10-1 M HCl/100 mM NaCl were titrated with aliquots of

0.4 M KOH, and UV-Vis spectra were recorded 2 min after each addition of base. The

122

protonation state of the chromophores was deduced from spectral changes around

isosbestic points. pKa´s were determined graphically from plots of pH vs.

log[k(Aacr/AacrH+)] (k = εacrH

+/εacr) using suitable absorbance maxima. Reported pKa´s are

means of three measurements. The apparent pKa had to be extrapolated to the pKa in

water [179].

The pKa determination of compound 60 was done by NMR measurements on a

Bruker DRX-500 MHz spectrometer. Compound 60 (7 mg) in 550 μL D2O was acidified

to pH 2 by adding 5 M DCl to the NMR tube, and an 1H NMR spectrum was recorded

each time after adding a small amount of NaOD solution, until the pH had reached 12.

The pKa value was determined graphically from a plot of pH vs. proton chemical shift δ.

Cytotoxicity Assay. The cytotoxicity studies were carried out using the Celltiter 96

Aqueous Non-Radioactive Cell Proliferation Assay kit. HL-60 leukemia cells and H460

lung cancer cells were kept in a humidified atmosphere of 5% CO2 at 37 oC. Cells were

plated on 96-well plates with a total 700 cells/well for H460 and 27,500 cells/well for

HL-60 cells. Stock solution of 57 was prepared in DMSO while the stock solutions of 58

and 60 were prepared in phosphate-buffered saline (PBS). Prior to the incubation, the

solution of 58 was diluted with media to a final concentration of less than 0.1% in DMSO.

The cells growing in log phase were incubated with appropriate serial dilutions of the

drugs in duplicate for 72 h. To each well was added 20 μL of MTS/PMS solution, and the

mixtures were allowed to equilibrate for 3 h. The absorbance at 490 nm was recorded

using a plate recorder (none of the derivatives tested absorbs at this wavelength). The


response equation in GraphPad Prism and are averages of two individual experiments.

123


4.3.1 Synthesis of Acridine-4-Carboxamides

The new derivatives were synthesized from the common precursor, N-(2-

(methylamino)ethyl)acridine-4-carboxamide (56) (Figure 58) [175, 177]. Transformation

of the secondary amino group into thiourea was accomplished by reacting 56 with

methylisothiocyanate to yield derivative 57 in 70–80% yield. The corresponding

platinum complex 58 was synthesized using the conjugation chemistry involving ligand

substitution in the activated precursor complex, [Pt(en)(DMF)Cl]+, to form a Pt–Sthiourea

linkage [100]. Complex 58 was isolated in its fully protonated form as the dinitrate salt.

The guanidine analogue, 60, was generated using guanidinylation chemistry developed

for the synthesis of compound 38. The reaction involves condensation of precursor

acridine-amine 56 with Boc-activated guanidylating reagent 45 in the presence of HgCl2,

followed by removal of the Boc group under mildly acidic conditions [178]. The product

was purified on basic alumina to afford 60 as the mono-HCl salt (Figure 58).

124

Figure 58. Synthesis of acridine-4-carboxamides 57 and 60 and platinum conjugate 58. Reagents and conditions: (i) MeNCS/EtOH, reflux; (ii) 1- [Pt(en)Cl2]/AgNO3, DMF, r.t., 2- 1M HNO3; (iii) HgCl2/ Et3N, DMF, 0 oC; (iv) 1- 1 M HCl, r.t., 2- Al2O3.

4.3.2 Acid–Base Chemistry

A major difference exists in the protonation states and overall charges between

the 4- and 9-substituted acridines. For the 9-amino derivatives, 37 and 38, the acridine is

fully protonated at physiological pH (pKa ~ 9–10) [100, 178], leading to + and 2+ overall

charges (pKa > 12 for the guanidinium moiety in 38, Chapter 2) [178]. In contrast, a

dramatic decrease in acridine basicity is observed for the newly synthesized 4-

carboxamides, for which pKa values in the range 3–4 were determined, consistent with

data reported previously for this class of compounds [171]. As a consequence, the 4-

substituted compounds carry a reduced positive charge and prove to be less soluble in

aqueous media than their 9-substituted counterparts. Electroneutral compound 57, for

instance, had to be dissolved in DMSO prior to serial dilution with PBS buffer in cell

proliferation assays.

N

O NH

NHN

S

1 23456

7 8 9

1011

12 13

1415

17

1819

20

21

22

23

24 2516

NH

O NH

NHN

SPt

ClH2N NH2

2+

(NO3)2

1 23

4567 8 9

1718

19

21

24 25

26 27

28 29

N

O NH

N

1 23

4567 8 9

1718

19

21

24 25

HN

NH2

ClN

O NH

N N

NH

O

O

i ii

iii

iv

N

O NH

HN

NH

NH

S

O

O

+

59 60

56 57

45

58

125

4.3.3 Cytotoxicities

Compounds 57, 58, and 60 were tested for their biological activities in a human

leukemia (HL-60) and a non-small cell lung cancer (NCI-H460) cell line (Table 6) using

a cell proliferation assay. Of the two new acridine-4-carboxamides, only the guanidine

derivative 60 showed appreciable cytotoxicity in HL-60 cells, where it proved to be more

active, by an order of magnitude, than its 9-substituted congener, 38. All other 4-

substituted derivatives were significantly less active than the 9-substituted derivatives,

and both 4- and 9- substituted compounds performed better in the solid tumor cell line. A

striking cytotoxic enhancement by >10-fold is observed for the hybrid agent 58 compared

to 57 in the NCI-H460 cell line. A similar trend has been observed for the prototypical 9-

substituted pair 37/31 (Table 6).

Table 6. Acid–Base Properties and In Vitro Cytotoxicity (IC50, μM) of Acridines and Platinum Acridines in HL-60 and NCI-H460 Cell Lines. Compound pKa1, pKa2

a Charge HL-60b NCI-H460 37 9.8 + 11.5 9.5 31 (not determined) 2+ 2.8 0.26 38 8.9, >12 2+ 79.7 10.3 57 3.4 0 >100 30.9 58 (not determined) + 16.0 2.4 60 2.7, >12 + 8.4 >100

a Spectrophotometric and 1H NMR spectroscopic pH titrations. pKa1 and pKa2 reflect the protonation of the acridine and guanidine moieties, respectively. b Concentrations needed to inhibit proliferation of HL-60 leukemia cancer cells and H460 lung cancer cells by 50%; average of two experiments.

The results of the cell proliferation assay suggest that the 4-substituted derivatives

are less potent than the 9-aminoacridines. Only monobasic compound 60 has an

126

advantage over dibasic compound 38 in HL-60 cells. This observation and the fact that

the relative enhancement of activity observed for the platinum conjugates compared to

the free ligands is less pronounced in the leukemia cell line than in the solid tumor

suggest that reduced uptake of the more highly charged drugs may limit their activity in

HL-60 cells. Derivative 57 was the least active compound in this series, probably due to

its poor aqueous solubility. Characteristically, the dual intercalating/platinating agents 31

and 58 showed the lowest IC50 values in H460 cells, which corroborates the view that

irreversible DNA adducts formed by the conjugates´ metal moieties contribute

considerably to the cell kill mechanism.

4.4 Conclusions

In the present study, acridine-4-carboxamide analogues of the previously studied

DNA-targeted thiourea- and guanidine-modified 9-aminoacridines were prepared. The

thiourea-based derivative (57) was introduced as a carrier ligand in the corresponding

platinum–intercalator conjugate (58). Differences in the DNA affinities (charge) and

binding modes between the 4- and 9-substituted compounds can be predicted. High-

resolution structural studies have been reported, which indicate critical differences in the

DNA binding modes of these agents. While in the DNA complexes of both types of

acridines the long dimension of the chromophore and the adjacent DNA base pairs are

co-aligned, differences exist in the directionality of intercalation: side chains in the 4-

position usually reside in the major groove, whereas 9-substituents preferentially protrude

into the minor groove [109, 114, 180]. ACRAMTU´s (37) distinct groove specificity of

intercalation has been implicated as the driving force in the platination of minor groove

127

sites by PT-ACRAMTU (31). Likewise, the preferred geometry of intercalation of

derivative 57 can be expected to affect the base and groove preference of platinum–DNA

adduct formation by conjugate 58. Thus, major differences can be predicted between the

DNA damage profiles of this hybrid agent and that of PT-ACRAMTU (31). It is

noteworthy to mention that precedent exists for this groove-specific reactivity in the

DNA interactions of analogous dual intercalating/alkylating agents: nitrogen mustard

groups attached to the 4-carboxamide residue alkylate guanine-N7 in the major groove,

whereas the major target of the analogous 9-anilino-linked mustards is adenine-N3 in the

minor groove [181]. It was rationalized that the minor groove adducts formed by PT-

ACRAMTU are intercalator-driven and contribute significantly to its cytotoxic effect

[10]. Finally, differences may exist in the recognition and repair of the DNA adducts

formed by 31 and 58. Taken together, the 4-substituted analogues do not appear to have

any advantage over the 9-substituted prototype, and the strategy of tuning its reactivity,

pursued in Chapter 3, turns out to be far superior to changing the linkage geometry of the

hybrids.

128

CHAPTER 5

SUMMARY AND OUTLOOK

129

The research in this dissertation was concerned with establishing structure–

activity relationships in a novel class of DNA-targeted platinum antitumor agents with

the ultimate goal of improving the biological activity of the prototypical agent, PT-

ACRAMTU (31). Two ways were explored of tuning the reactivity and target

interactions of platinum, which was considered critical in changing the pharmacological

properties of this pharmacophore. The first approach (Chapters 2 and 3) involved a

structurally minimally invasive modification of the prototype to enhance its chemical

stability and reactivity with DNA. This was achieved by introducing a new inert

nonleaving group in place of thiourea, or, in other words, by simply changing the ligand

donor set of the platinum moiety from [PtN3S] to [PtN4]. In the second approach

(Chapter 4), an acridine-4-carboxamide derivative of PT-ACRAMTU was generated, in

which the point of attachment of the platinum-modified linker was changed from the 9-

position to the 4-position of the planar chromophore. It was reasoned that this change in

geometry might have consequences for the DNA groove and sequence selectivity of

platinum and, ultimately, the cytotoxicity of the adducts formed.

To investigate the effect of sulfur on the substitution chemistry of platinum in

reactions with nucleobase nitrogen and potential consequences of DNA damage profile of

the conjugate, a nitrogen analogue of ACRAMTU, 1-[2-(acridine-9-ylamino)ethyl]-1,3-

dimethylguanidine (38) was synthesized in which thiourea-S is replaced with guanidine-

NH. While this study was able to delineate unusual pathways to novel cyclic and

spirocyclic acridine derivatives, the ultimate goal of generating a guanidine analogue of

PT-ACRAMTU was not reached due to the chelating properties of the guanidinato group.

Instead, two all-nitrogen derivatives of PT-ACRAMTU containing an amidine-NH

130

linkage (54 and 55), based on the ligand N-(2-(acridin-9-ylamino)ethyl)-N-

methylpropionimidamide, could be synthesized using platinum-mediated amidination

chemistry. A relationship was discovered between the rate of DNA binding and

cytotoxic potential: it was found that replacement of thiourea-S with amidine-NH, which

accelerates platinum binding to nucleobase nitrogen, leads to a dramatic increase in cell

toxicity. On the other hand, it was also demonstrated that the change in donor group

reduces the (undesired) reactivity of platinum with cysteine sulfur. These findings

suggest that the direct reaction of platinum with cysteine sulfur, a strong nucleophile, is

controlled by the cis-effect. The fact that the opposite scenario is observed for DNA

nitrogen supports the view that steric effects dominate this reaction, which most likely

requires hydrolytic activation (aquation) of platinum. To test if the increased reactivity of

compound 54 with DNA nitrogen leads to higher nuclear levels of platinum in cancer

cells, a cellular imaging study using confocal fluorescence microscopy was performed.

The study revealed higher intracellular levels of complex 54 than of PT-ACRAMTU.

Unfortunately, direct detection of platinum in the nucleus was complicated by DNA-

mediated quenching of acridine fluorescence. A high concentration of drug appears to be

located to the lysosomes. The biological relevance of this effect, which is most likely a

consequence of the protonation state of acridine in a low-pH environment, is unclear.

Both amidine-modified complexes, 54 and 55, show nanomolar cytotoxicity in H460

cells, and derivative 55 is able to slow the growth of tumors in the corresponding

xenograft model. The concentrations of platinum detected in various biological tissues

reveal that, while compound 55 reaches the tumors, the majority of platinum accumulated

131

in the kidneys, the major organ of excretion, which most likely contributes to the toxicity

observed in the treated animals.

Unlike the amidine-based derivatives of PT-ACRAMTU, 54 and 55, the

compound’s 4-carboxamide analogue, [PtCl(en)(N-(2-(1,3-

dimethylthioureido)ethyl)acridine-4-carboxamide)]NO3 (58), did not offer any

improvement compared to the prototype. It may be speculated that the adducts formed

by this complex are less cytotoxic or more easily repaired than those formed by PT-

ACRAMTU. Other factors, such as reduced DNA binding because of the lower charge

on the complex due to the lowered pKa of the acridine carrier, may also contribute to the

relatively low activity of compound 58.

In conclusion, the research in this dissertation has performed two different

structure-activity relationship studies that will have an impact on the future development

of these platinum-based therapies into treatments for incurable cancers. In particular, the

new amidine-based analogues represent a new lead in the race for a cure of aggressive,

chemoresistant cancers, such as NSCLC. As with most cytotoxic agents, one problem

that remains to be solved is their high systemic toxicity, which, in the present case, is

most likely the result of the promiscuous reactivity of platinum with biological

nucleophiles. The future design of analogues of compounds 54 and 55 should therefore

be concerned with improving the therapeutic index of this type of agent. One strategy

might be to reduce the reactivity of the metal with sulfur nucleophiles, which may be

possible by tuning the substitution chemistry of the current lead compounds. In addition,

targeting the new hybrids specifically to cancer cells may be another option of enhancing

the pharmacological properties of these agents.

132

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Appendix A

Figure A1. 1H NMR (top) and 13C-{1H} NMR (bottom) spectra of compound 38 in D2O.

NH

NHN

HNH2N

Cl

Cl

38

156


N

N

N

NH Cl

40

157

Figure A3. 1H NMR (top) and 13C-{1H} NMR (bottom) spectra of compound 42 in DMF-d7.

NH

N

N

N

NN

N

42

158


N

N

N

NH2 Br

43

159

Figure A5. 1H NMR (top) and 13C-{1H} NMR (bottom) spectra of compound 45 in CDCl3.

NH

NH

S

O

O

45

160

Figure A6. 1H NMR (top) and 13C-{1H} NMR (bottom) spectra of compound 46 in CDCl3.

NH

N

S

O

O

O

O

46

161

Figure A7. 1H NMR (top) and 13C-{1H} NMR (bottom) spectra of compound 47 in MeOH-d4.

N

NHN

HNNO

O

47

162

Figure A8. 1H NMR (top) and 13C-{1H} NMR (bottom) spectra of compound 52 in D2O..

PtH2N NH2

Cl N

+

Cl

52

163

Figure A9. 1H NMR spectrum of compound 52´ in MeOH-d4.

PtH2N NH2

Cl N

+

Cl

15 15

52´

164


PtH3N NH3

Cl N

+

Cl

53

165


NH

NH

NHNPt

H2N NH2

Cl(NO3)2

2+

-

54

166

Figure A12. 1H NMR spectrum of compound 54´ in DMF-d7.

NH

NH

NHNPt

H2N NH2

Cl(NO3)2

2+

-15 15

54´

167


NH

NH

NHNPt

H3N NH3

Cl(NO3)2

2+

-

55

168

Figure A14. 1H NMR (top) and 13C-{1H} NMR (bottom) spectra of compound 57 in DMSO-d6.

N

O NH

NHN

S

57

169


NH

O NH

NHN

SPt

ClH2N NH2

(NO3)2

2+

58

170

Figure A16. 1H NMR spectrum of compound 59 in MeOH-d4.

N

O NH

N N

NH

O

O

59

171


N

O NH

NHN

NH2

Cl

60

172

Appendix B Table B1. Summary of X-ray Crystallographic Data for 1-[2-(Acridine-9-ylamino)-

ethyl]-1,3-dimethylguanidine Dihydrochloride (ACRAMGU·2HCl), 38.

Empirical formula C37H50 N14 O13

Formula weight 898.91

Temperature (K) 193(2)

gCrystal system Triclinic

Space group P1 - C1i (No. 2)

a (Å) 8.1233(17)

b (Å) 16.267(4)

c (Å) 17.002(4)

α (°) 109.081(4)

β (°) 100.725(4)

γ (°) 94.547(4)

V (Å3) 2062.3(8)

Z 2

dcalc (g cm-3) 1.448

Absorption coefficient (mm-1) 0.112 mm-1

Crystal size (mm3) 0.13 × 0.11 × 0.05

θ range (°) 3.79-25.00

Reflections collected 15189

Unique reflections 7216 [R(int) = 0.0652]

Data obsvd (I>2σI), parameters 7216 / 626

Goodness-of-fit on F2 0.869

R1, wR2 (for I>2σI)a,b 0.0560, 0.0950

R1, wR2 (all data) 0.1057, 0.1088

Largest diff. peak and hole (e Å-3) 0.477, -0.285 aR1 = Σ|| Fo|-|Fc||/Σ|Fo| bwR2 = [Σ[w(Fo

2-Fc2)2]/Σw(Fo

2)2]1/2

173

Table B2. Bond Lengths [Å] and Angles [°] for 1-[2-(Acridine-9-ylamino)-ethyl]-1,3-

dimethylguanidine Dihydrochloride (ACRAMGU·2HCl), 38.

N(1)-C(1) 1.328(3) N(4A)-C(14A) 1.334(4) N(1)-C(16) 1.458(3) N(4A)-C(18A) 1.443(4) N(1)-H(1N) 0.79(3) N(4A)-H(4NA) 0.87(3) N(2)-C(3) 1.361(3) N(5A)-C(14A) 1.328(4) N(2)-C(4) 1.362(3) N(5A)-H(5N3) 0.94(3) N(2)-H(2N) 0.87(3) N(5A)-H(5N4) 0.87(3) N(3)-C(14) 1.336(3) C(1A)-C(2A) 1.441(4) N(3)-C(15) 1.462(3) C(1A)-C(5A) 1.451(4) N(3)-C(17) 1.466(3) C(2A)-C(3A) 1.408(4) N(4)-C(14) 1.325(4) C(2A)-C(6A) 1.430(4) N(4)-C(18) 1.462(4) C(3A)-C(9A) 1.407(4) N(4)-H(4N) 0.84(3) C(4A)-C(5A) 1.398(4) N(5)-C(14) 1.322(4) C(4A)-C(10A) 1.411(4) N(5)-H(5N1) 0.93(3) C(5A)-C(13A) 1.410(4) N(5)-H(5N2) 0.91(3) C(6A)-C(7A) 1.356(4) C(1)-C(2) 1.440(4) C(7A)-C(8A) 1.401(4) C(1)-C(5) 1.448(4) C(8A)-C(9A) 1.361(4) C(2)-C(3) 1.423(4) C(10A)-C(11A) 1.365(4) C(2)-C(6) 1.427(4) C(11A)-C(12A) 1.394(4) C(3)-C(9) 1.405(4) C(12A)-C(13A) 1.358(4) C(4)-C(10) 1.400(4) C(15A)-C(16A) 1.504(4) C(4)-C(5) 1.407(4) N(6)-O(3) 1.243(3) C(5)-C(13) 1.412(4) N(6)-O(1) 1.245(3) C(6)-C(7) 1.366(4) N(6)-O(2) 1.255(3) C(7)-C(8) 1.392(4) N(7)-O(5) 1.237(3) C(8)-C(9) 1.360(4) N(7)-O(6) 1.245(3) C(10)-C(11) 1.364(4) N(7)-O(4) 1.264(3) C(11)-C(12) 1.396(4) N(8)-O(9) 1.228(3) C(12)-C(13) 1.367(4) N(8)-O(7) 1.248(3) C(15)-C(16) 1.511(4) N(8)-O(8) 1.245(3) N(1A)-C(1A) 1.328(3) N(9)-O(11) 1.211(4) N(1A)-C(16A) 1.469(3) N(9)-O(12) 1.213(4) N(1A)-H(1NA) 0.81(2) N(9)-O(10) 1.241(4) N(2A)-C(4A) 1.358(4) O(1S)-C(1S) 1.405(5) N(2A)-C(3A) 1.363(3) O(1S)-H(1S) 0.95(6) N(2A)-H(2NA) 0.91(3) N(3A)-C(15A) 1.463(3) N(3A)-C(14A) 1.333(4) N(3A)-C(17A) 1.461(4)

174

Table B2, Continued N(1)-C(1)-C(2) 124.7(3) N(1)-C(1)-C(5) 116.8(3) C(1)-N(1)-C(16) 130.1(3) C(2)-C(1)-C(5) 118.4(3) C(1)-N(1)-H(1N) 118(2) C(3)-C(2)-C(6) 115.9(3) C(16)-N(1)-H(1N) 112(2) C(3)-C(2)-C(1) 118.2(3) C(3)-N(2)-C(4) 123.4(3) C(6)-C(2)-C(1) 125.9(3) C(3)-N(2)-H(2N) 118.6(18) N(2)-C(3)-C(9) 118.2(3) C(4)-N(2)-H(2N) 118.0(17) N(2)-C(3)-C(2) 120.3(3) C(14)-N(3)-C(15) 121.3(2) C(9)-C(3)-C(2) 121.5(3) C(14)-N(3)-C(17) 119.7(2) N(2)-C(4)-C(10) 119.3(3) C(15)-N(3)-C(17) 116.7(2) N(2)-C(4)-C(5) 119.6(3) C(14)-N(4)-C(18) 123.9(3) C(10)-C(4)-C(5) 121.1(3) C(14)-N(4)-H(4N) 125(2) C(4)-C(5)-C(13) 117.4(3) C(18)-N(4)-H(4N) 111(2) C(4)-C(5)-C(1) 119.5(3) C(14)-N(5)-H(5N1) 120.4(18) C(13)-C(5)-C(1) 123.1(3) C(14)-N(5)-H(5N2) 121.9(19) C(7)-C(6)-C(2) 121.8(3) H(5N1)-N(5)-H(5N2) 118(3) C(6)-C(7)-C(8) 120.3(3) C(1A)-N(1A)-C(16A) 131.0(3) C(9)-C(8)-C(7) 121.0(3) C(1A)-N(1A)-H(1NA) 115(2) C(8)-C(9)-C(3) 119.6(3) C(16A)-N(1A)-H(1NA) 113(2) C(11)-C(10)-C(4) 119.5(3) C(4A)-N(2A)-C(3A) 122.4(3) C(10)-C(11)-C(12) 120.7(3) C(4A)-N(2A)-H(2NA) 119.9(18) C(13)-C(12)-C(11) 120.2(3) C(3A)-N(2A)-H(2NA) 117.4(18) C(12)-C(13)-C(5) 121.1(3) C(14A)-N(3A)-C(17A) 120.9(2) N(5)-C(14)-N(4) 119.0(3) C(14A)-N(3A)-C(15A) 121.7(3) N(5)-C(14)-N(3) 119.9(3) C(17A)-N(3A)-C(15A) 116.6(2) N(4)-C(14)-N(3) 121.0(3) C(14A)-N(4A)-C(18A) 122.9(3) N(1A)-C(1A)-C(2A) 124.4(3) C(14A)-N(4A)-H(4NA) 117(2) N(1A)-C(1A)-C(5A) 117.0(3) C(18A)-N(4A)-H(4NA) 120(2) C(2A)-C(1A)-C(5A) 118.6(3) C(14A)-N(5A)-H(5N3) 119(2) C(3A)-C(2A)-C(6A) 115.8(3) C(14A)-N(5A)-H(5N4) 120(2) C(3A)-C(2A)-C(1A) 118.2(3) H(5N3)-N(5A)-H(5N4) 120(3) C(6A)-C(2A)-C(1A) 126.0(3) N(3)-C(15)-C(16) 111.0(2) N(2A)-C(3A)-C(2A) 121.1(3) N(1)-C(16)-C(15) 110.2(2) N(2A)-C(3A)-C(9A) 117.1(3) N(3A)-C(15A)-C(16A) 110.5(2) C(2A)-C(3A)-C(9A) 121.8(3) N(1A)-C(16A)-C(15A) 109.2(2) N(2A)-C(4A)-C(5A) 120.6(3) O(3)-N(6)-O(1) 120.9(3) N(2A)-C(4A)-C(10A) 118.4(3) O(3)-N(6)-O(2) 118.8(3) C(5A)-C(4A)-C(10A) 121.0(3) O(1)-N(6)-O(2) 120.2(2) C(4A)-C(5A)-C(13A) 117.2(3) O(5)-N(7)-O(6) 120.8(3) C(4A)-C(5A)-C(1A) 119.0(3)

175

Table B2, Contd. O(5)-N(7)-O(4) 120.3(3) C(13A)-C(5A)-C(1A) 123.8(3) O(6)-N(7)-O(4) 119.0(2) C(7A)-C(6A)-C(2A) 121.5(3) O(9)-N(8)-O(7) 120.0(3) C(6A)-C(7A)-C(8A) 121.2(3) O(9)-N(8)-O(8) 120.6(3) C(9A)-C(8A)-C(7A) 119.5(3) O(7)-N(8)-O(8) 119.2(3) C(8A)-C(9A)-C(3A) 120.0(3) O(11)-N(9)-O(12) 121.0(4) C(11A)-C(10A)-C(4A) 119.3(3) O(11)-N(9)-O(10) 119.4(5) C(10A)-C(11A)-C(12A) 120.6(3) O(12)-N(9)-O(10) 119.5(5) C(13A)-C(12A)-C(11A) 120.1(3) C(1S)-O(1S)-H(1S) 108(3) C(12A)-C(13A)-C(5A) 121.8(3) N(5A)-C(14A)-N(4A) 118.7(3) N(5A)-C(14A)-N(3A) 120.1(3) N(3A)-C(14A)-N(4A) 121.2(3)

176

Table B3. Summary of X-ray Crystallographic Data for (Z)-N-[1-(Acridin-9-yl)-3-

methylimidazolidin-2-ylidene]methanaminium Hydrochloride, 40.

Empirical formula C18H19 N4Cl



Crystal system Orthorhombic

Space group Pbca – D152h (No. 61)

a (Å) 11.5196(15)

b (Å) 11.3782(16)

c (Å) 23.977(3)

V (Å3) 3142.7(7)

Z 8



Crystal size (mm3) 0.47 × 0.10 × 0.09

θ range (°) 1.70-26.40





R1, wR2 (for I>2σI)a,b 0.0448, 0.1016

R1, wR2 (all data) 0.0666, 0.1086


2-Fc2)2]/Σw(Fo

2)2]1/2

177

Table B4. Bond Lengths [Å] and Angles [°] for (Z)-N-[1-(Acridin-9-yl)-3-methylimidazolidin-2-ylidene]methanaminium Hydrochloride, 40. N(1)-C(14) 1.358(2) C(1)-C(5) 1.392(3) N(2)-C(3) 1.345(2) C(1)-C(2) 1.396(3) N(2)-C(4) 1.346(2) C(2)-C(6) 1.426(3) N(3)-C(14) 1.325(2) C(2)-C(3) 1.438(3) N(4)-C(14) 1.321(2) C(3)-C(9) 1.435(3) N(1)-C(1) 1.441(2) C(4)-C(10) 1.426(3) N(1)-C(16) 1.482(2) C(4)-C(5) 1.434(3) N(3)-C(17) 1.449(2) C(5)-C(13) 1.428(3) N(3)-C(15) 1.462(2) C(6)-C(7) 1.365(3) N(4)-C(18) 1.462(2) C(7)-C(8) 1.420(3) N(4)-H(4N) 0.90(2) C(8)-C(9) 1.359(3) C(15)-C(16) 1.519(3) C(10)-C(11) 1.352(3) C(12)-C(13) 1.357(3) C(11)-C(12) 1.411(3) C(14)-N(1)-C(1) 123.40(16) N(2)-C(4)-C(10) 118.32(18) C(14)-N(1)-C(16) 108.66(16) N(2)-C(4)-C(5) 122.98(18) C(1)-N(1)-C(16) 119.19(15) C(10)-C(4)-C(5) 118.70(18) C(3)-N(2)-C(4) 118.33(17) C(1)-C(5)-C(13) 123.92(17) C(14)-N(3)-C(17) 126.36(17) C(1)-C(5)-C(4) 117.51(17) C(14)-N(3)-C(15) 111.62(16) C(13)-C(5)-C(4) 118.50(18) C(17)-N(3)-C(15) 121.13(16) C(7)-C(6)-C(2) 121.08(19) C(14)-N(4)-C(18) 124.82(17) C(6)-C(7)-C(8) 120.0(2) C(14)-N(4)-H(4N) 119.8(14) C(9)-C(8)-C(7) 121.3(2) C(18)-N(4)-H(4N) 113.3(14) C(8)-C(9)-C(3) 120.24(19) C(5)-C(1)-C(2) 120.74(17) C(11)-C(10)-C(4) 120.45(19) C(5)-C(1)-N(1) 119.56(17) C(10)-C(11)-C(12) 121.1(2) C(2)-C(1)-N(1) 119.64(17) C(13)-C(12)-C(11) 120.6(2) C(1)-C(2)-C(6) 124.26(18) C(12)-C(13)-C(5) 120.57(19) C(1)-C(2)-C(3) 117.14(17) N(4)-C(14)-N(3) 123.84(18) C(6)-C(2)-C(3) 118.60(18) N(4)-C(14)-N(1) 124.56(18) N(2)-C(3)-C(9) 118.14(18) N(3)-C(14)-N(1) 111.57(17) N(2)-C(3)-C(2) 123.15(18) N(3)-C(15)-C(16) 102.55(16) C(9)-C(3)-C(2) 118.70(18) N(1)-C(16)-C(15) 104.31(16)

178

Table B5. Summary of X-ray Crystallographic Data for 2′-(1H-Imidazol-1-yl)-8′-methyl-

7′,8′-dihydro-6′H,10H-spiro-[acridine-9,4′-imidazo[1,2-a][1,3,5]triazine], 42.

Empirical formula C21H19 N7



Crystal system Monoclinic

Space group P2(1)/c - C 52h (No. 14)

a (Å) 9.030(9)

b (Å) 11.573(12)

c (Å) 16.754(17)

V (Å3) 1745(3)

Z 4



Crystal size (mm3) 0.27 × 0.12 × 0.12

θ range (°) 3.89-25.03





R1, wR2 (for I>2σI)a,b 0.0512, 0.1172

R1, wR2 (all data) 0.0723, 0.1243


2-Fc2)2]/Σw(Fo

2)2]1/2

179

Table B6. Bond Lengths [Å] and Angles [°] for 2′-(1H-Imidazol-1-yl)-8′-methyl-7′,8′-

dihydro-6′H,10H-spiro-[acridine-9,4′-imidazo[1,2-a][1,3,5]triazine], 42.

N(1)-C(3) 1.380(3) N(7)-C(20) 1.370(3) N(1)-C(4) 1.392(3) C(1)-C(2) 1.513(3) N(1)-H(1N) 0.99(3) C(1)-C(5) 1.523(3) N(2)-C(14) 1.351(3) C(2)-C(3) 1.389(3) N(2)-C(16) 1.468(3) C(2)-C(6) 1.390(3) N(2)-C(1) 1.494(3) C(3)-C(9) 1.394(3) N(3)-C(14) 1.337(3) C(4)-C(10) 1.390(3) N(3)-C(17) 1.446(3) C(4)-C(5) 1.395(3) N(3)-C(15) 1.455(3) C(5)-C(13) 1.394(3) N(4)-C(14) 1.314(3) C(6)-C(7) 1.371(3) N(4)-C(18) 1.373(3) C(7)-C(8) 1.381(3) N(5)-C(18) 1.276(3) C(8)-C(9) 1.373(3) N(5)-C(1) 1.457(3) C(10)-C(11) 1.366(3) N(6)-C(19) 1.362(3) C(11)-C(12) 1.380(3) N(6)-C(21) 1.375(3) C(12)-C(13) 1.371(3) N(6)-C(18) 1.424(3) C(15)-C(16) 1.523(3) N(7)-C(19) 1.308(3) C(20)-C(21) 1.345(3) C(3)-N(1)-C(4) 119.5(2) C(2)-C(3)-C(9) 120.1(2) C(3)-N(1)-H(1N) 122.2(14) C(10)-C(4)-N(1) 119.6(2) C(4)-N(1)-H(1N) 117.5(14) C(10)-C(4)-C(5) 120.3(2) C(14)-N(2)-C(16) 109.84(18) N(1)-C(4)-C(5) 120.0(2) C(14)-N(2)-C(1) 117.94(17) C(13)-C(5)-C(4) 118.3(2) C(16)-N(2)-C(1) 122.97(16) C(13)-C(5)-C(1) 122.48(19) C(14)-N(3)-C(17) 124.49(19) C(4)-C(5)-C(1) 119.16(19) C(14)-N(3)-C(15) 110.71(17) C(7)-C(6)-C(2) 120.9(2) C(17)-N(3)-C(15) 122.00(18) C(6)-C(7)-C(8) 119.7(2) C(14)-N(4)-C(18) 110.65(18) C(9)-C(8)-C(7) 120.7(2) C(18)-N(5)-C(1) 117.11(18) C(8)-C(9)-C(3) 119.6(2) C(19)-N(6)-C(21) 106.35(18) C(11)-C(10)-C(4) 119.9(2) C(19)-N(6)-C(18) 126.83(18) C(10)-C(11)-C(12) 120.6(2) C(21)-N(6)-C(18) 126.82(17) C(13)-C(12)-C(11) 119.7(2) C(19)-N(7)-C(20) 104.76(19) C(12)-C(13)-C(5) 121.1(2) N(5)-C(1)-N(2) 108.35(15) N(4)-C(14)-N(3) 124.74(19) N(5)-C(1)-C(2) 110.61(17) N(4)-C(14)-N(2) 125.0(2) N(2)-C(1)-C(2) 107.25(16) N(3)-C(14)-N(2) 110.19(19) N(5)-C(1)-C(5) 111.14(17) N(3)-C(15)-C(16) 102.47(17) N(2)-C(1)-C(5) 110.31(17) N(2)-C(16)-C(15) 102.03(18)

180

Table B6, Contd. C(2)-C(1)-C(5) 109.11(18) N(5)-C(18)-N(4) 131.64(19) C(3)-C(2)-C(6) 118.9(2) N(5)-C(18)-N(6) 116.32(19) C(3)-C(2)-C(1) 119.7(2) N(4)-C(18)-N(6) 112.03(17) C(6)-C(2)-C(1) 121.4(2) N(7)-C(19)-N(6) 112.0(2) N(1)-C(3)-C(2) 120.3(2) C(21)-C(20)-N(7) 111.25(19) N(1)-C(3)-C(9) 119.5(2) C(20)-C(21)-N(6) 105.68(19)

181

Table B7. Summary of X-ray Crystallographic Data for 1-(Acridin-9-yl)-3-methylimidazolidin-2-imine Hydrobromide, 43.

Empirical formula C17H17BrN4




Space group Cc – C 4s (No. 9)

a (Å) 11.124(1)

b (Å) 20.633(2)

c (Å) 7.1005(8)

V (Å3) 1519.8(3)

Z 4



Crystal size (mm3) 0.23 × 0.16 × 0.04

θ range (°) 4.21-30.48



Data obsvd (I>2σI), restraints, parameters 4117 / 2 / 208


R1, wR2 (for I>2σI)a,b 0.0484, 0.1171

R1, wR2 (all data) 0.0511, 0.1193


2-Fc2)2]/Σw(Fo

2)2]1/2

182

Table B8. Bond Lengths [Å] and Angles [°] for 1-(Acridin-9-yl)-3-methyl-imidazolidin-2-imine Hydrobromide, 43. N(1)-C(13) 1.342(4) C(3)-C(4) 1.418(6) N(1)-C(1) 1.346(4) C(4)-C(5) 1.365(5) N(2)-C(14) 1.356(4) C(5)-C(6) 1.425(4) N(2)-C(7) 1.415(4) C(6)-C(7) 1.400(4) N(2)-C(16) 1.479(4) C(7)-C(8) 1.402(4) N(3)-C(14) 1.331(4) C(8)-C(9) 1.428(4) N(3)-C(17) 1.448(5) C(8)-C(13) 1.431(4) N(3)-C(15) 1.459(5) C(9)-C(10) 1.364(4) N(4)-C(14) 1.319(4) C(10)-C(11) 1.419(5) C(1)-C(2) 1.423(4) C(11)-C(12) 1.355(5) C(1)-C(6) 1.433(4) C(12)-C(13) 1.432(4) C(2)-C(3) 1.362(5) C(15)-C(16) 1.534(5) C(13)-N(1)-C(1) 118.6(2) C(6)-C(7)-N(2) 118.4(3) C(14)-N(2)-C(7) 125.6(2) C(8)-C(7)-N(2) 121.4(2) C(14)-N(2)-C(16) 110.5(3) C(7)-C(8)-C(9) 123.6(3) C(7)-N(2)-C(16) 121.4(2) C(7)-C(8)-C(13) 117.5(2) C(14)-N(3)-C(17) 125.1(3) C(9)-C(8)-C(13) 118.9(3) C(14)-N(3)-C(15) 111.4(3) C(10)-C(9)-C(8) 120.2(3) C(17)-N(3)-C(15) 119.2(3) C(9)-C(10)-C(11) 120.8(3) N(1)-C(1)-C(2) 118.6(3) C(12)-C(11)-C(10) 120.9(3) N(1)-C(1)-C(6) 122.8(2) C(11)-C(12)-C(13) 120.4(3) C(2)-C(1)-C(6) 118.6(3) N(1)-C(13)-C(8) 123.2(3) C(3)-C(2)-C(1) 120.7(3) N(1)-C(13)-C(12) 118.0(3) C(2)-C(3)-C(4) 120.3(3) C(8)-C(13)-C(12) 118.8(3) C(5)-C(4)-C(3) 121.5(3) N(4)-C(14)-N(3) 126.2(3) C(4)-C(5)-C(6) 119.4(3) N(4)-C(14)-N(2) 123.3(3) C(7)-C(6)-C(5) 122.6(3) N(3)-C(14)-N(2) 110.5(3) C(7)-C(6)-C(1) 117.8(2) N(3)-C(15)-C(16) 103.1(3) C(5)-C(6)-C(1) 119.6(3) N(2)-C(16)-C(15) 102.3(3) C(6)-C(7)-C(8) 120.1(3)

183

Table B9. Summary of X-ray Crystallographic Data for [Pt(EtCN)(C19H22N4)Cl2], 50.

Empirical formula C22H27Cl2N5Pt




Space group P21/c - C 52h (No. 14)

a (Å) 13.791(2)

b (Å) 12.107(1)

c (Å) 14.747(2)

V (Å3) 2062.3(8)

Z 4



Crystal size (mm3) 0.16 × 0.12 × 0.07

θ range (°) 3.79-27.50





R1, wR2 (for I>2σI)a,b 0.0432, 0.0942

R1, wR2 (all data) 0.0662, 0.1032

Largest diff. peak and hole (e Å-3) 2.672, -1.104

aR1 = Σ|| Fo|-|Fc||/Σ|Fo| bwR2 = [Σ[w(Fo

2-Fc2)2]/Σw(Fo

2)2]1/2

184

Table B10. Bond Lengths [Å] and Angles [°] for [Pt(EtCN)(C19H22N4)Cl2], 50. Pt(1)-N(1) 1.989(5) C(4)-C(16) 1.468(8) Pt(1)-N(5) 1.993(5) C(4)-C(5) 1.486(8) Pt(1)-Cl(1) 2.299(2) C(5)-C(6) 1.397(9) Pt(1)-Cl(2) 2.290(2) C(5)-C(10) 1.403(8) N(1)-C(1) 1.291(7) C(6)-C(7) 1.385(9) N(2)-C(1) 1.338(8) C(7)-C(8) 1.389(10) N(3)-C(4) 1.284(7) C(8)-C(9) 1.347(11) N(4)-C(11) 1.373(8) C(9)-C(10) 1.393(8) N(4)-C(10) 1.383(8) C(11)-C(12) 1.385(9) N(2)-C(2) 1.463(8) C(11)-C(16) 1.419(8) N(2)-C(19) 1.470(8) C(12)-C(13) 1.364(10) N(3)-C(3) 1.434(7) C(13)-C(14) 1.377(9) N(1)-H(1N) 0.90(6) C(14)-C(15) 1.389(9) N(4)-H(4N) 0.74(2) C(15)-C(16) 1.401(8) C(1)-C(17) 1.512(9) N(5)-C(20) 1.110(8) C(2)-C(3) 1.514(9) C(20)-C(21) 1.471(10) C(17)-C(18) 1.509(10) C(21)-C(22) 1.510(10) N(1)-Pt(1)-N(5) 178.2(2) N(3)-C(4)-C(5) 117.3(5) N(1)-Pt(1)-Cl(2) 90.31(15) C(16)-C(4)-C(5) 114.7(5) N(5)-Pt(1)-Cl(2) 90.73(17) C(6)-C(5)-C(10) 118.0(6) N(1)-Pt(1)-Cl(1) 88.79(15) C(6)-C(5)-C(4) 121.8(6) N(5)-Pt(1)-Cl(1) 90.13(17) C(10)-C(5)-C(4) 120.2(6) Cl(2)-Pt(1)-Cl(1) 178.59(6) C(7)-C(6)-C(5) 120.9(6) C(1)-N(1)-Pt(1) 129.6(5) C(6)-C(7)-C(8) 119.8(7) C(1)-N(1)-H(1N) 113(4) C(9)-C(8)-C(7) 120.2(7) Pt(1)-N(1)-H(1N) 117(4) C(8)-C(9)-C(10) 121.2(7) C(1)-N(2)-C(2) 121.4(5) N(4)-C(10)-C(9) 121.0(6) C(1)-N(2)-C(19) 124.0(5) N(4)-C(10)-C(5) 119.0(6) C(2)-N(2)-C(19) 114.6(5) C(9)-C(10)-C(5) 120.0(6) C(4)-N(3)-C(3) 123.8(5) N(4)-C(11)-C(12) 120.4(6) C(11)-N(4)-C(10) 123.2(5) N(4)-C(11)-C(16) 119.2(6) C(11)-N(4)-H(4N) 119(4) C(12)-C(11)-C(16) 120.4(6) C(10)-N(4)-H(4N) 117(4) C(13)-C(12)-C(11) 121.1(6) N(2)-C(2)-C(3) 115.6(5) C(12)-C(13)-C(14) 120.2(6) N(3)-C(3)-C(2) 111.3(5) C(13)-C(14)-C(15) 119.2(6) C(18)-C(17)-C(1) 109.7(6) C(14)-C(15)-C(16) 122.3(6) C(20)-C(21)-C(22) 112.7(6) C(15)-C(16)-C(11) 116.1(6) N(1)-C(1)-N(2) 120.5(6) C(15)-C(16)-C(4) 124.1(6)

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Table B10, Contd. N(1)-C(1)-C(17) 120.4(6) C(11)-C(16)-C(4) 119.7(6) N(2)-C(1)-C(17) 118.9(5) C(20)-N(5)-Pt(1) 177.1(6) N(3)-C(4)-C(16) 127.9(6) N(5)-C(20)-C(21) 178.0(8)

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Table B11. Summary of X-ray Crystallographic Data for [PtCl(en)(C19H23N4)](NO3)2, 54.

Empirical formula C21H31ClN8O6Pt



gCrystal system Monoclinic

Space group C2/c

a (Å) 34.070(7)

b (Å) 10.887(2)

c (Å) 14.023(3)

V (Å3) 5071.7(18)

Z 8



Crystal size (mm3) 0.13 × 0.11 × 0.05

θ range (°) 3.79-25.00





R1, wR2 (for I>2σI)a,b 0.0456, 0.0834

R1, wR2 (all data) 0.0790, 0.0933


2-Fc2)2]/Σw(Fo

2)2]1/2

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Table B12. Bond Lengths [Å] and Angles [°] for [PtCl(en)(C19H23N4)](NO3)2, 54. Pt(1)-N(1) 2.023(3) C(4)-C(16) 1.453(11) Pt(1)-N(5) 2.025(7) C(5)-C(6) 1.410(11) Pt(1)-N(6) 2.013(7) C(5)-C(10) 1.431(12) Pt(1)-Cl(1) 2.313(2) C(6)-C(7) 1.355(11) N(1)-C(1) 1.276(9) C(7)-C(8) 1.382(12) N(2)-C(1) 1.371(8) C(8)-C(9) 1.361(11) N(3)-C(4) 1.320(9) C(9)-C(10) 1.411(11) N(4)-C(10) 1.339(9) C(11)-C(16) 1.404(11) N(4)-C(11) 1.352(9) C(11)-C(12) 1.412(11) N(2)-C(2) 1.450(9) C(12)-C(13) 1.314(12) N(2)-C(19) 1.460(9) C(13)-C(14) 1.402(13) N(3)-C(3) 1.479(8) C(14)-C(15) 1.364(11) N(5)-C(20) 1.475(10) C(15)-C(16) 1.413(11) N(6)-C(21) 1.469(11) N(7)-O(1) 1.255(11) C(1)-C(17) 1.506(11) N(7)-O(2) 1.225(9) C(2)-C(3) 1.524(11) N(7)-O(3) 1.189(10) C(17)-C(18) 1.543(12) N(8)-O(4) 1.209(10) C(20)-C(21) 1.463(13) N(8)-O(5) 1.233(10) C(4)-C(5) 1.453(11) N(8)-O(6) 1.227(10)

N(6)-Pt(1)-N(1) 174.2(2) C(8)-C(9)-C(10) 120.3(9) N(6)-Pt(1)-N(5) 83.3(3) N(4)-C(10)-C(9) 119.0(8) N(1)-Pt(1)-N(5) 90.9(2) N(4)-C(10)-C(5) 121.3(7) N(6)-Pt(1)-Cl(1) 90.5(2) C(9)-C(10)-C(5) 119.7(8) N(1)-Pt(1)-Cl(1) 95.32(12) N(4)-C(11)-C(16) 120.1(8) N(5)-Pt(1)-Cl(1) 173.5(2) N(4)-C(11)-C(12) 120.6(8) C(1)-N(1)-Pt(1) 129.6(4) C(16)-C(11)-C(12) 119.3(8) C(1)-N(2)-C(2) 123.0(6) C(13)-C(12)-C(11) 121.3(9) C(1)-N(2)-C(19) 117.4(5) C(12)-C(13)-C(14) 121.0(8) C(2)-N(2)-C(19) 118.5(5) C(15)-C(14)-C(13) 119.7(9) C(4)-N(3)-C(3) 132.4(6) C(14)-C(15)-C(16) 120.8(8) C(10)-N(4)-C(11) 123.4(6) C(11)-C(16)-C(15) 117.8(8) C(20)-N(5)-Pt(1) 109.4(5) C(11)-C(16)-C(4) 119.5(8) C(21)-N(6)-Pt(1) 110.6(6) C(15)-C(16)-C(4) 122.6(8) N(1)-C(1)-N(2) 122.4(7) N(2)-C(2)-C(3) 113.7(6) N(1)-C(1)-C(17) 119.3(7) N(3)-C(3)-C(2) 108.9(6) N(2)-C(1)-C(17) 118.3(7) C(1)-C(17)-C(18) 113.9(7) N(3)-C(4)-C(5) 125.6(7) C(21)-C(20)-N(5) 110.7(8) N(3)-C(4)-C(16) 116.5(7) C(20)-C(21)-N(6) 108.5(8)

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Table B12, Contd. C(5)-C(4)-C(16) 117.8(7) O(3)-N(7)-O(2) 122.4(10) C(6)-C(5)-C(10) 116.8(7) O(3)-N(7)-O(1) 120.3(11) C(6)-C(5)-C(4) 125.8(8) O(2)-N(7)-O(1) 117.3(10) C(10)-C(5)-C(4) 117.4(7) O(4)-N(8)-O(6) 121.5(9) C(7)-C(6)-C(5) 121.9(9) O(4)-N(8)-O(5) 119.9(9) C(6)-C(7)-C(8) 120.7(9) O(6)-N(8)-O(5) 118.6(9) C(9)-C(8)-C(7) 120.5(8)

189

Appendix C Table C1. Platinum Concentrations in Various Biological Tissues (Tumors, Kidneys and Lungs) at Two Different Doses (0.1 mg/kg and 0.5 mg/kg) Determined by ICP–MS After 16 Hours of Digestion.

Dose Sample I.D. Tumor (ppb) Kidney (ppb) Lung (ppb) Control 8788 0.001 0 0

8384 0.004 0 0.001 6970 0.002 0 0 6768 0.003 0 0.001 5758 0 0.001 0

0.1mg/kg 8586 0.368 4.838 0.321 6364 0.101 8.202 0.206 6162 0.506 12.04 0.313 5556 0.205 9.803 0.234 5354 0.494 7.895 0.228

0.5mg/kg 8990 0.018 7.761 0.225 8182 0.279 5.797 0.151 7374 0.426 6.653 0.169 7172 0.349 4.979 0.33 5152 0.063 4.869 0.218

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Table C2. Masses of Various Biological Tissues (Tumors, Kidneys and Lungs).

Sample I.D. Tumor (g) Kidney (g) Lung (g) 8788 0.5817 0.4100 0.1342 8384 1.1586 0.3477 0.1883 6970 0.6836 0.2881 0.1942 6768 0.6548 0.3083 0.1355 5758 0.7600 0.2972 0.1410

8586 0.5419 0.1494 0.1808 6364 0.1731 0.2917 0.1354 6162 0.6317 0.3029 0.1643 5556 0.2606 0.3181 0.1510 5354 0.6965 0.3120 0.1259

8990 0.0368 0.3091 0.1430 8182 0.6270 0.2901 0.2202 7374 0.5221 0.2272 0.1032 7172 0.7529 0.2940 0.1763 5152 0.1410 0.2720 0.1512

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SCHOLASTIC VITA

Zhidong Ma

BORN: September 15, 1982 Xining, Qinghai, P. R. China UNDERGRADUATE Nankai University STUDY: Tianjin, P. R. China B.Sc., Chemistry, 2004 GRADUATE STUDY: Wake Forest University Winston-Salem, North Carolina, USA Ph.D. candidate, 2009 SCHOLASTIC AND PROFESSIONAL EXPERIENCE: Research Assistant, Wake Forest University 2005-2007 Winston-Salem, North Carolina, USA

Teaching Assistant, Wake Forest University 2004-2005, 2007-2009 Winston-Salem, North Carolina, USA HONORS AND AWARDS: American Institute of Chemists Outstanding Graduate Student Award 2009 Richter Scholar Award 2009 1st-runner up prize for poster presentation at the 2008 Wake Forest University Graduate Student Research Day 2008 Graduate Student Oral Presentation Award at the 2008 Cancer Biology Department Research Retreat 2008 WFU Alumni Student Travel Award 2007, 2008

192

PUBLICATIONS: Z. Ma, L. Rao and U. Bierbach: Replacement of a Thiourea-S with an Amidine-NH Donor Group in a Platinum-Amidine Antitumor Compound Reduces the Metal’s Reactivity with Cysteine Sulfur. Journal of Medicinal Chemistry, 52, 3424. (2009) Z. Ma, J. Roy Choudhury, M. W. Wright, C. S. Day, G. Saluta, G. L. Kucera and U. Bierbach: A Non-Cross-Linking Platinum-Acridine Agent with Potent Activity in Non-Small-Cell Lung Cancer. Journal of Medicinal Chemistry, 51, 7574. (2008) Z. Ma, G. Saluta, G. L. Kucera and U. Bierbach: Effect of linkage geometry on biological activity in thiourea- and guanidine- substituted acridines and platinum-acridines. Bioorganic & Medicinal Chemistry Letters, 18, 3799. (2008) Z. Ma, C. S. Day and U. Bierbach: Unexpected Reactivity of the 9-Aminoacridine Chromophore in Guanidylation Reactions. Journal of Organic Chemistry, 72, 5387. (2007) PRESENTATIONS: Z. Ma and U.Bierbach: A Platinum-Acridine Agent with Activity Against Non-Small Cell Lung Cancer (NSCLC) In Vitro and In Vivo, Presented at the 60th Southeast Regional Meeting of the American Chemical Society, Nashville, TN, November 12-15, 2008. (Oral Presentation) Z. Ma, J. Roy Choudhury, G. Saluta, G. L. Kucera and Ulrich Bierbach: Tuning the reactivity and DNA interactions of platinum-acridine hybrid agents. Presented at the 59th Southeast Regional Meeting of the American Chemical Society, Greenville, SC, October 24-27, 2007. (Poster) Z. Ma, C. S. Day and U. Bierbach: Tuning the DNA Binding of Cytotoxic Platinum-Acridine Conjugates. Presented at the 3rd Asian Biological Inorganic Chemistry Conference (AsBIC III), Nanjing, China, October 30-November 3, 2006. (Poster) Z. Ma, C. S. Day and U. Bierbach: Synthesis and Characterization of DNA-Targeted Acridinylguanidines. Presented at the 57th Southeast Regional Meeting of the American Chemical Society, Memphis, TN, November 1-4, 2005. (Poster)

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