Thymidine Kinase As a Molecular Target for The

Home , Deoxynucleoside kinase, Thymidine kinase 1, Thymidine monophosphate

THYMIDINE KINASE AS A MOLECULAR TARGET FOR THE

DEVELOPMENT OF NOVEL ANTICANCER AND ANTIBIOTIC AGENTS

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

Youngjoo Byun, M.S.

*****

The Ohio State University 2006

Dissertation Committee: Approved by

Professor Werner Tjarks, Advisor

Professor Robert W. Curley, Jr. Advisor Professor Pui-Kai Li Graduate Program in Pharmacy ABSTRACT

The purpose of this dissertation is to develop novel boron-delivery agents for the treatment of glioblastoma multiforme (GBM) and to discover novel antibiotics for the treatment of anthrax infections.

GBM is one of the most aggressive of all cancers. Despite significant advances in the treatment of almost all other types of cancer, the survival of patients diagnosed with

GBM has remained almost unchanged for decades. The GBM accounts for approximately

50% of newly diagnosed primary brain tumors and its yearly incidence in the USA is 4.3 cases per 100,000 people. Bacillus anthracis is a Gram-positive, spore-forming, anaerobic bacterium that causes anthrax. Due to the infectiousness of B. anthracis spores and the high mortality of inhalational anthrax, the major concern with anthrax in the 21st century is its use as a biological weapon. Therefore, both GBM and anthrax infections are major public health concerns in USA. Thymidine kinase (TK), a key enzyme of the salvage pathway for DNA biosynthesis, is an attractive molecular target for both diseases because of its high activity in tumor cells and its frequent occurrence in pathogenic bacteria. The goal of our research is to develop TK-targeting anticancer agents for GBM and TK-targeting antibiotics for B. anthracis.

In GBM-related research, boron neutron capture therapy (BNCT) was chosen as the therapeutic modality. BNCT kills tumor cells without damaging normal brain tissue ii by utilizing a nuclear reaction of the boron-10 isotope (10B) with thermal neutrons.

Previous structure-activity relationship (SAR) studies of 3-carboranyl thymidine

analogues (3CTAs) identified one promising boron-delivery agent for BNCT, designated

N5-2OH. Two feasible synthetic routes for the preparation of 10B-enriched N5-2OH were

developed and it was evaluated as a boron-delivery agent for BNCT in pilot neutron

irradiation experiments. Treatment of rats bearing intracerebral F98 glioma with 10B- enriched N5-2OH and neutron irradiation resulted in prolonged survival compared with control groups, indicating that 10B-enriched N5-2OH has a significant potential as a

boron-delivery agent for BNCT of GBM. Stereochemical- and geometrical N5-2OH

isomers were synthesized and their substrate specificities for human thymidine kinase 1

(hTK1) were evaluated to complete SAR studies of the N5-2OH series. A computational

model for the binding of the N5-2OH series to the active site of hTK1 was also developed.

In order to improve physicochemical properties of 3CTAs, zwitterionic nido

3CTAs were synthesized and evaluated as boron-delivery agents for BNCT. All

compounds of this 3CTA subclass were good substrates for hTK1. In in vivo studies, one

zwitterionic nido 3CTA accumulated selectively in mice implanted with TK1-expressing

L929 (wt) tumors versus those implanted with L929 TK1 (-) tumors. In addition, it showed improved water solubility compared with N5-2OH. The zwitterionic nido m-

carborane system constituted a novel boron-carrying moiety for BNCT drug development,

and thus, its unique properties were further investigated. The zwitterionic nido m- carborane cluster exhibited unexpected chemical features by reacting with the carbonyl group of ketones or aldehydes without addition of acid catalyst to afford zwitterionic iminum-substituted carborane.

iii In anthrax-related research, a compound library composed of known antiviral and

anticancer drugs was screened in two assay systems to identify a lead compound for the

development of novel anthrax-specific antibiotics. Phosphoryl transfer assays showed

that most of the tested thymidine analogues of this library were good substrates of both

TK from B. anthracis (BaTK) and hTK1. However, only FLT and Floxuridine showed

activity in in vitro toxicity studies with B. anthracis Sterne.

Due to the limited success in identifying a lead compound out of a library of

known antiviral and anticancer drugs, twenty thymidine analogues were designed and

synthesized as potential inhibitors of BaTK and thymidine monophosphate kinase of B. anthracis (BaTMPK), which is another key enzyme in DNA biosynthesis. The inhibitors were designed to mimic thymidine triphosphate (dTTP), the endogenous feedback inhibitor of BaTK. The phosphate moiety of dTTP was replaced with sulfonamide, amide, urea, thiourea, or triazole groups in these inhibitors. Some of the urea-, thiourea-, and triazole-linked inhibitors exhibited relatively low IC50 values in preliminary growth

inhibition studies with B. anthracis Sterne. Overall, the concept of novel antibiotics

targeting BaTK and/or BaTMPK was validated and two inhibitors were selected as lead

compounds for further structural optimization.

Dedicated to Mom and Seonghee

v ACKNOWLEDGMENTS

It is difficult to overstate my gratitude to my advisor, Dr. Werner Tjarks. Without his inspiration and guidance, this thesis would not be possible. Throughout my graduate

research period from 2002 through 2006, he has provided encouragement, limitless

advice, good teaching, and lots of good ideas for me. I would have been lost without him.

Also, I would like to thank the faculty of the Division of Medicinal Chemistry and

Pharmacognosy in College of Pharmacy for their teaching and encouragement. In

particular, I thank my committee members, Drs. Robert W. Curley, Jr. and Pui-Kai Li for

their helpful guidance and valuable advice.

I greatly appreciate Kathy Brooks and John Fowble for making my graduate

school life much easier.

I wish to express my thanks to my collaborators, Dr. Staffan Eriksson at the

Swedish University of Agricultural Sciences in Uppsala, Sweden, Dr. Rolf F. Barth at the

OSU Department of Pathology, and Dr. Andrew Phipps at the OSU Department of

Veterinary Biosciences for carrying out in vitro and in vivo experiments.

I am grateful to my former and current lab members for their help in numerous matters. Especially, I would like to mention Dr. Jayaseharan Johnsamuel for comprehensive instructions in computational chemistry and Rohit Tiwari for his detailed

vi critique on this thesis. Special thanks also go to Dr. B.T.S. Thirumamagal, Andrew

Schaub, and Susan Vogel for their collaboration.

For financial support in form of fellowships, I thank the Graduate School of The

Ohio State University and The Procter & Gamble Company.

I remain indebted to my Mom, my parents-in-law, and many family members in

South Korea for their support and encouragement.

Last, but most importantly, I would like to dedicate this thesis to my wife

Seonghee and my sons Wonjae and Hyunjae for their love and patience. Without your sacrifices and support, I could not have made my dreams come true.

vii

VITA

June 22, 1970……………………………Born – Kyunggi, South Korea

1994……………………………………. B.S. Pharmacy, Seoul National University,

Seoul, South Korea

1994-1996……………………………….M.S. Pharmacy, Seoul National University

1996-2001……………………………….Research Scientist,

Drug Discovery and Development

Amorepacific Corp., Seoul, South Korea

2001-2006……………………………….Graduate Teaching and Research Associate,

The Ohio State University

PUBLICATIONS

(1) Y. Byun; S. Narayanasamy; J. Johnsamuel; A. K. Bandyopadhyaya; R. Tiwari; A. S. Al-Madhoun; R. F. Barth; S. Eriksson; W. Tjarks. 3-Carboranyl thymidine analogues (3CTAs) and other boronated nucleosides for boron neutron capture therapy. Anti-Cancer Agents in Medicinal Chemistry. 2006, 6, 127-144.

(2) Y. Byun; J. Yan; A. S. Al-Madhoun; J. Johnsamuel; W. Yang; R. F. Barth; S. Eriksson; W. Tjarks. Synthesis and biological evaluation of neutral and zwitterionic 3-carboranyl thymidine analogues for boron neutron capture therapy. Journal of Medicinal Chemistry. 2005, 48, 1188-1198.

viii

(3) Y. Byun; J. Yan; A. S. Al-Madhoun; J. Johnsamuel; W. Yang; R. F. Barth; S. Eriksson; W. Tjarks The synthesis and biochemical evaluation of thymidine analogues substituted with nido carborane at the N-3 position. Applied Radiation and Isotopes. 2004, 61, 1125-1130.

(4) S. S. Shin; Y. Byun; K. M. Lim; J. K. Choi; K.-W. Lee; J. H. Moh; J. K. Kim; Y. S. Jeong; J. Y. Kim; Y. H. Choi; H.-J. Koh; Y.-H. Park; Y. I. Oh; M.-S. Noh; S. Chung. In vitro structure-activity relationship and in vivo studies for a novel class of cyclooxygenase-2 inhibitors: 5-aryl-2,2-dialkyl-4-phenyl-3(2H)furanone derivatives. Journal of Medicinal Chemistry. 2004, 47, 792-804.

(5) R. F. Barth; W. Yang; A. S. Al-Madhoun; J. Johnsamuel; Y. Byun; S. Chandra; D. R. Smith; W. Tjarks; S. Eriksson. Boron-containing nucleosides as potential delivery agents for neutron capture therapy of brain tumors. Cancer Research. 2004, 64, 6287-6295.

(6) J. Johnsamuel; N. Lakhi; A. S. Al-Madhoun; Y. Byun; J. Yan; S. Eriksson; W. Tjarks. Synthesis of ethyleneoxide modified 3-carboranyl thymidine analogues and evaluation of their biochemical, physicochemical, and structural properties. Bioorganic & Medicinal Chemistry. 2004, 12, 4769-4781.

(7) J. Johnsamuel; Y. Byun; T. P. Jones; Y. Endo; W. Tjarks. A new strategy for molecular modeling and receptor-based design of carborane containing compounds. Journal of Organometallic Chemistry. 2003, 680, 223-231.

(8) J. Johnsamuel; Y. Byun; T. P. Jones; Y. Endo; W. Tjarks. A convenient method for the computer-aided molecular design of carborane containing compounds. Bioorganic & Medicinal Chemistry Letters. 2003, 13, 3213-3216.

(9) K.-W. Lee; Y. H. Choi; Y. H. Joo; J. K. Kim; S. S. Shin; Y. Byun; Y. Kim; S. Chung. A facile one-pot synthesis of 4,5-diaryl-2,2-dimethyl-3(2H)-furanones. Bioorganic & Medicinal Chemistry. 2002, 10, 1137-1142.

(10) S. S. Shin; M. S. Noh; Y. Byun; J. K. Choi; J. Y. Kim; K. M. Lim; J. Y. Ha; J. K. Kim; C. H. Lee; S. Chung. 2,2-Dimethyl-4,5-diaryl-3(2H)furanone derivatives as selective cyclo-oxygenase-2 inhibitors. Bioorganic & Medicinal Chemistry Letters. 2001, 11, 165-168.

FIELD OF STUDY

Major Field: Pharmacy

Specialization: Medicinal Chemistry & Pharmacognosy

TABLE OF CONTENTS

Page

Abstract...... ii

Dedication...... v

Acknowledgments...... vi

Vita...... viii

List of tables...... xv

List of figures...... xvi

List of schemes ...... xviii

Abbreviations...... xix

Chapters:

1. Thymidine kinase as a molecular target for anticancer, antiviral, and antibiotic therapy……...... 1

1.1 Introduction...... 1 1.2 De novo pathway versus salvage pathway of dNTPs ...... 1 1.3 dNKs (Deoxyribonucleoside kinases)...... 3 1.4 Crystal structures of dNKs...... 6 1.5 Clinical implications of dNKs ...... 9 1.6 Human thymidine kinase 1 (hTK1) ...... 11 1.6.1 hTK1 and TK2...... 11 1.6.2 Biochemical properties of hTK1...... 12 1.7 TK1 and drug design...... 13

2. Boron neutron capture therapy as a modality to treat glioblastoma multiforme .. 14

2.1 Glioblastoma multiforme (GBM) ...... 14 2.2 Boron neutron capture therapy (BNCT) ...... 16 2.2.1 Principles...... 16 2.2.2 Keys to success of BNCT ...... 16 2.2.3 Boron-carrying agents in clinical trials...... 17 2.3 Boronated thymidine analogues as BNCT agents ...... 18 2.3.1 Boron-containing cellular building blocks...... 18 2.3.2 Biochemical metabolic pathway of boron-containing thymidine analogues...... 19 2.3.3 hTK1 and brain tumors ...... 21 2.3.4 Evaluation of hTK1 substrate characteristics of boron-containing dThds in phosphorylation transfer assay (PTA) ...... 22 2.4 Carboranes as boron moieties of BNCT agents...... 23 2.4.1 Carborane structures...... 23 2.4.2 Preparation of carboranes...... 24 2.4.3 The chemistry of carboranes...... 25 2.4.4 Theoretical calculations of carboranes...... 26 2.5 Carborane-containing dThds...... 27 2.5.1 dThds substituted with a carborane cage at the carbohydrate protion...... 27 2.5.2 dThds substituted with a carborane cage at the C-5 position...... 27 2.5.3 dThds substituted with a carborane cage at the N-3 position (3CTAs) ...... 28

3. 10B-enriched N5-2OH: A promising boron-delivery agent for BNCT ...... 30

3.1 Background and rationale ...... 30 3.2 Preparation and analysis of 10B-enriched N5-2OH ...... 32 3.2.1 Synthesis...... 32 3.2.2 Analysis...... 37 3.3 Preparation and analysis of pure N5-2OH epimers and geometrical isomers ...... 39 3.3.1 Synthesis...... 39 3.3.2 Analysis...... 42 3.4 In silico studies ...... 46 3.5 Biological evaluation...... 51 3.5.1 Phosphoryl transfer assays (PTAs) ...... 51 3.5.2 In vivo studies ...... 52 3.5.2 Neutron irradiation studies...... 53 3.6 Summary and conclusions ...... 55 3.7 Outlook: Challenges ahead ...... 55

xii

4. Investigation of biochemical, biological, and chemical properties of zwitterionic nido carborane ...... 58

4.1 Background and rationale ...... 58 4.2 Synthesis and biological evaluation of zwitterionic nido 3CTAs for BNCT ...... 59 4.2.1 Synthesis...... 59 4.2.2 Analysis...... 61 4.2.3 Phosphoryl transfer assays (PTAs) ...... 64 4.2.4 In vivo studies ...... 67 4.2.5 Summary and conclusions...... 69 4.3 Experimental and theoretical studies of zwitterionic nido m-carborane system ...... 70 4.3.1 Synthesis...... 70 4.3.2 Structural analysis...... 71 4.3.3 Theoretical calculations...... 74 4.3.4 Observations...... 77 4.3.5 Applications of novel zwitterionic nido m-carborane cluster...... 81 4.3.6 Summary and conclusions...... 82 4.4 Outlook...... 83

5. Discovery of novel antibiotics targeting thymidine kinase and thymidine monophosphate kinase from Bacillus anthracis...... 85

5.1 Introduction...... 85 5.2 Rationale...... 86 5.3 Evaluation of agents with established molecular targets in mammalian and viral systems as potential antibiotics against B. anthracis...... 88 5.3.1 BaTK and hTK1 PRs ...... 93 5.3.2 Molecular modeling...... 93 5.3.3 In vitro toxicity assay with B. anthracis Sterne...... 95 5.4 BaTK- and/or BaTMPK-specific inhibitors...... 98 5.4.1 Drug design...... 98 5.4.2 Synthesis...... 101 5.4.3 Biological evaluation...... 105 5.4.4 Molecular modeling...... 108 5.4.4.1 B. anthracis thymidine kinase (BaTK) ...... 108 5.4.4.2 B. anthracis thymidine monophosphate kinase ...... 109 5.5 Summary and conclusions ...... 112 5.6 Outlook ...... 113

6. Summary and conclusions ...... 114

7. Experimental section ...... 118

xiii

7.1 General methods ...... 118 7.2 Chapter 3 ...... 120 7.3 Chapter 4 ...... 149 7.4 Chapter 5 ...... 175 7.5 Phosphoryl transfer assays (PTAs) ...... 194 7.6 In vitro toxicity studies ...... 196 7.7 In vivo uptake experiments (Chapter 4)...... 197 7.8 Preclinical BNCT experiments (Chapter 3)...... 197 7.9 Molecular modeling...... 198 7.9.1 Homology modeling...... 198 7.9.2 Theoretical calculations using Gaussian 03 program ...... 199 7.9.3 Calculations of polar surface areas (PSAs) and apolar surface areas (APSAs) ...... 199 7.9.4 Docking studies using the FlexX module in Sybyl 7.1...... 200

Bibliography ...... 201

Appendix A 1H-NMR and 13C-NMR spectra of 3.5.A ...... 232

xiv

LIST OF TABLES

Table Page

1.1 Protein sequence identities of dNKs using BLAST 2.2.6...... 5

1.2 Structural information of dNKs deposited in Protein Data Bank ...... 8

2.1 TK1 and dCK activities in brain tumor cell lines and normal brain...... 22

3.1 Summary of biological data of N5-2OH...... 31

3.2 hTK1 PRs, retention times, and ratios of apolar to polar surface are (APSA/PSA) of the N5-2OH isomers...... 45

3.3 Boron distribution in F98-glioma-bearing rats at 1 and 2.5 h after administration of BPA and N5-2OH by CED...... 53

3.4 Survival of F98-glioma-berating rats in preclinical irradiation studies...... 54

4.1 hTK1/TK2 PRs and RP-18 retention times of 3CTAs, dThd, and AZT...... 62

4.2 Tumor uptake of 4.15.B, N5-2OH, and BPA in mice bearing s.c. implants of L929 (wt) and L929 TK1 (-) tumors ...... 68

4.3 Total energies of optimized structures 4.21 and 4.22 ...... 74

4.4 Mulliken- and APT charges of heavy atoms and the bridging hydrogen atom for 4.21 and 4.22 at the B3LYP/6-31G** level...... 75

4.5 Experimental and theoretical (GIAO) 11B-NMR chemical shifts of 4.22 ...... 76

5.1 PRs and in vitro growth inhibition of the existing compound library ...... 91-92

5.2 In vitro IC50 of dThd analogues against B.anthracis Sterne ...... 107

LIST OF FIGURES

Figure Page

1.1 Structure of 2´-deoxyribonucleoside triphosphates (dNTPs) ...... 2

1.2 De novo and salvage pathways for dNTP synthesis ...... 3

1.3 Crystal structures of dNKs...... 10

2.1 Nuclear capture and fission reactions by 10B and thermal neutron...... 16

2.2 BNCT agents in clinical trials...... 17

2.3 Biochemical salvage pathway of boron-containing thymidines ...... 20

2.4 5´-Monophosphorylation of dThd or boronated dThds by hTK1...... 23

2.5 Closo- and nido structures of o-, m-, and p-carborane...... 24

2.6 Potential dThd-based BNCT agents in preclinical trials...... 28

3.1 By-products observed in steps a, b, and c of Scheme 3.2...... 35

3.2 ESI-mass spectra of compounds 3.5.A and 3.5.B...... 37

3.3 2D-HMBC spectrum of compound 3.5.A...... 38

3.4 Partial 1H-NMR spectra of 3.11.A, 3.11.B, 3.11.C, and 3.5.A with (S)-(+)-2,2,2- trifluoro-1-(9-anthryl)-ethanol in CDCl3 at 300 K...... 43

3.5 HPLC spectrum of a mixture of geometrical N5-2OH isomers (3.11.C, 3.11.D, and 3.11.E) using water/acetonitrile gradient ...... 44

3.6 The optimized structures for 3.11.A, (R)-epimers of 3.11.D and 3.11.E at B3LYP/6-31G* level using Gaussian 03 program ...... 47

3.7 (A) hTK1 crystal structure (PDB ID: 1W4R) with dTTP...... 49 xvi

(B) hTK1 homology model docked with 3.11.A ...... 49 (C) hTK1 homology model docked with the (R)-epimer of 3.11.D ...... 49 (D) hTK1 homology model docked with the (R)-epimer of 3.11.E...... 49

4.1 Partial 1H-NMR spectra of two pure epimers of 4.14.B...... 63

4.2 Phosphorylation of neutral closo-carboranyl 3CTAs 4.15.A, 4.17.A, and N5-2OH, anionic nido-carboranyl 3CTA 4.20, zwitterionic nido-carboranyl 3CTAs 4.15.B and 4.17.B, and dThd by recombinant hTK1...... 65

4.3 Structures of 4.21 and 4.22 optimized at the B3LYP/6-31G* level ...... 71

4.4 11B (proton-decoupled)-11B (proton-decoupled) COSY spectrum of 4.22 ...... 72

4.5 Proton-decoupled 11B-NMR spectra of 4.22 in deuterated acetone solution recorded as a function of time...... 77

4.6 UV absorption spectra of 4.22 and 4.23 ...... 79

4.7 Diagram of HOMO and LUMO for 4.22, 4.23, and benzene...... 80

4.8 Chemical structures of 2nd generation 3CTAs ...... 83

5.1 Salvage and de novo biochemical pathways for dTTP synthesis ...... 87

5.2 Chemical structures of the existing compound library ...... 90

5.3 Dimension of dTTP binding pocket in hTK1 crystal structure (A) and the BaTK homology model (B)...... 94

5.4 Known antibiotics substituted with phosphate mimics at the 5´-position ...... 99

5.5 Design of phosphate-mimicking dThd analogues as BaTK and/or BaTMPK inhibitors ...... 100

5.6 The hTK1 crystal structure with dTTP (A) and the BaTK homology model docked with 5.11 (B) and 5.21 (C) ...... 108

5.7 (A) Crystal structure of human TMPK with dTMP and ADP...... 110 (B) Crystal structure of SaTMPK with dTMP...... 110 (C) BaTMPK homology model with 5.11 ...... 110 (D) BaTMPK homology model with 5.21 ...... 110 (E) BaTMPK homology model with 5.9...... 111 (F) Crystal structure of hTMPK with 5.11...... 111

xvii

LIST OF SCHEMES

Scheme Page

3.1 Synthesis of 11B-abundant N5-2OH by Al-Madhoun et al...... 33

3.2 Preparation of 10B-enriched N5-2OH from o-carborane ...... 34

3.3 Synthesis of 10B-enriched N5-2OH from decaborane ...... 36

3.4 Synthetic route for pure N5-2OH epimers...... 40

3.5 Synthetic route for geometrical N5-2OH isomers ...... 41

3.6 Iodination of N5-2OH...... 56

4.1 Synthesis of zwitterionic nido 3CTAs and the neutral closo counterparts ...... 60

4.2 Synthesis of zwitterionic nido m-carborane (4.22)...... 70

4.3 Preparation of iminium-containing compounds (4.23-4.25) from 4.22...... 78

4.4 Plausible mechanism of formation of 4.23 from 4.22 with acetone ...... 82

5.1 Synthesis of 5´-amino-5´-deoxythymidine as a key intermediate ...... 101

5.2 Synthesis of sulfonamide-linked dThd inhibitors...... 102

5.3 Synthesis of thiourea- and urea-linked dThd inhibitors...... 103

5.4 Synthesis of amide-linked dThd inhibitors...... 104

5.5 Synthesis of triazole-linked dThd inhibitors...... 105

xviii

LIST OF ABBREVIATIONS

Abbreviation Term

°C Degrees in Celsius 1D 1-Dimensional 2D 2-Dimensional 3D 3-Dimensional 3CTAs 3-Carboranyl thymidine analogues 5-FU 5-Fluorouracil ADP Adenosine diphosphate ATP Adenosine triphosphate AZT 3´-Azido-3´-deoxythymidine 10B Boron-10 isotope 11B Boron-11 isotope B. anthracis Bacillus anthracis BaTK Bacillus anthracis thymidine kinase BaTMPK Bacillus anthracis thymidine monophosphate kinase BBB Blood-brain barrier BNCT Boron neutron capture therapy Boc Butoxycarbamate Bu Butyl c Concentration (g/100 mL) CaTK Clostridium acetobutylicum thymidine kinase CED Convection-enhanced delivery COSY Correlation spectroscopy Da Dalton

xix

DCP-AES Direct current plasma-atomic emission spectroscopy DEPT Distortionless enhancement of polarisation transfer dCK 2´-Deoxycytidine kinase dGK 2´-Deoxyguanosine kinase DMAP 4-(Dimethylamino)pyridine DMF N,N-Dimethylformamide DMSO Dimethyl sulfoxide DNA 2´-Deoxyribonucleic acid dNK Deoxyribonucleoside kinase dThd Thymidine dThds Thymidine analogues dTMP Thymidine monophosphate dTTP Thymidine triphosphate dUrd 2´-Deoxyuridine eq Equivalents ESI Eletrospray ionization Et Ethyl g Gram GBM Glioblastoma multiforme h Hour HMBC Heteronuclear multiple-bond correlation HMQC Heteronuclear multiple-quantum coherence HOMO Highest occupied molecular orbital HPLC High performance liquid chromatography HRMS High resolution mass spectroscopy hTK1 Human thymidine kinase 1 hTMPK human thymidine monophosphate kinase Hz Hertz

IC50 Inhibitory concentration 50% IR Infra-red

LDL Low-density lipoprotein LUMO Lowest occupied molecular orbital m Meta M Molar Me Methyl MHz Megahertz min Minute mol Mole MP Monophosphate N Normality n-BuLi n-Butyllithium NMO 4-Methyl morpholine N-oxide NMR Nuclear magnetic resonance o Ortho

OsO4 Osmium tetraoxide p Para ppm Parts per million PR Phosphorylation rate PTA Phosphoryl transfer assay RP-8 C8 reversed phase RP-18 C18 reversed phase rt Room temperature SAR Structure activity relationship SaTMPK Staphylococcus aureus thymidine monophosphate kinase TBAF Tetrabutylammonium fluoride TFA Trifluoroacetic acid THF Tetrahydrofuran TK1 Thymidine kinase 1 TK2 Thymidine kinase 2

xxi

TLC Thin layer chromatography TMPK Thymidine monophosphate kinase UuTK Ureaplasma urealyticum thymidine kinase UV Ultraviolet

xxii CHAPTER 1

THYMIDINE KINASE AS A MOLECULAR TARGET FOR

ANTICANCER, ANTIVIRAL, AND ANTIBIOTIC THERAPY

1.1. Introduction

DNA (2´-deoxyribonucleic acid) is a polynucleotide chain molecule that encodes

genetic information. DNA biosynthesis requires four types of 2´-deoxyribonucleoside triphosphates (dNTPs) as essential precursors, which are 2´-deoxyadenosine triphosphate

(dATP), 2´-deoxyguanosine triphosphate (dGTP), 2´-deoxycytidine triphosphate (dCTP), and thymidine triphosphate (dTTP). Each dNTP is composed of three subunits: a nitrogen-containing base, a five-carbon sugar, and three phosphate groups, as shown in

Figure 1.1. The nitrogen-containing bases are classified into two pyrimidine bases, thymine (T) and cytosine (C), and two purine bases, adenine (A) and guanine (G). The carbohydrate unit in DNA is 2´-deoxy-D-ribose while D-ribose is found in RNA.

1.2. De novo pathway versus salvage pathway of dNTPs

dNTPs are biosynthesized by two pathways: De novo pathway and salvage pathway,1,2 which are depicted in Figure 1.2. The de novo synthesis of dNTPs begins

with basic cellular building blocks such as glutamine, glycine, aspartate, 5-

1 phosphoribosyl-1-pyrophosphate, CO2 and folic acid. In case of purine-based dNTPs, inosine monophosphate (IMP) is the key intermediate while uridine monophosphate

(UMP) is the key component in pyrimidine-based dNTP biosynthesis. Ribonucleotide reductase (RR) is another key enzyme of the de novo pathway, which reduces ribose in all four ribonucleotides to 2´-deoxyribose.3

Triphosphate Sugar

O O O 5' Base -O P O P O P O O - - - 1' O O O 4' 3' 2' OH

O O NH2 NH2 NH N N Base: N N NH N O N N O N N N NH2 dTTP dCTP dATP dGTP

Figure 1.1. Structures of 2´-deoxyribonucleoside triphosphates (dNTPs). dTTP: Thymidine triphosphate, dCTP: 2´-deoxycytidine triphosphate, dATP: 2´-deoxyadenosine triphosphate, dGTP: 2´-deoxyguanosine triphosphate.

The salvage pathway utilizes 2´-deoxyribonucleosides (dNs) from intra- or extracellular pools generated from DNA catabolism.1 Deoxyribonucleoside kinases

(dNKs) catalyzes the initial transfer of the γ-phosphate from ATP to the 5´-hydroxyl group of dNs to form 2´-deoxyribonucleoside-5´-monophosphates (dNMPs).4 This step is generally considered to be the rate-limiting step in the dNK salvage pathway. The dNMPs are further phosphorylated to dNDPs (2´-deoxyribonucleoside-5´-diphosphates) by nucleoside monophosphate kinases (NMPKs). Finally, nucleoside diphosphate kinases

2 (NDPKs) catalyze the formation of dNTPs. The initial phosporylation by dNKs is

irreversible, while the following two steps catalysized by NMPKs and NDPKs are

reversible. Intracellularly, dephosphorylation of dNMPs occurs primarily via 5´- nucleotidases (5´-NTs).5

Figure 1.2. De novo and salvage pathways of dNTP synthesis

dN: 2´-deoxyribonucleosides, dNMP: 2´-deoxyribonucleoside monophosphates, dNDP: 2´- deoxyribonucleoside diphosphates, dNTP: 2´-deoxyribonucleoside triphosphates, NMP: nucleoside monophosphates, NDP: nucleoside diphosphates, dNKs: Deoxyribonucleoside kinase, NMPK: nucleoside monophosphate kinase, NDPK: nucleoside diphosphate kinase, RR: ribonucleotide reductase, 5´-NT: 5´- nucleotidase.

1.3. dNKs (Deoxyribonucleoside kinases)

Mammals have four distinct dNKs; thymidine kinase 1 (TK1), deoxycytidine

kinase (dCK), thymidine kinase 2 (TK2), and deoxyguanosine kinase (dGK).1 TK1 and

dCK are found in the cytosol while TK2 and dGK are located in the mitochondria. TK1 3 converts thymidine (dThd) and 2´-deoxyuridine (dUrd) to the corresponding

monophosphates (dTMP and dUMP) in the presence of ATP and Mg2+. Deoxycytidine

kinase (dCK) phosphorylates endogenous 2´-deoxycytidine (dCyd), 2´-deoxyguanosine

(dGuo), and 2´-deoxyadenosine (dAdo) to the corresponding monophosphates (dCMP,

dGMP, and dAMP, respectively). Mitochondrial TK2 phosphorylates pyrimidine-based

dNs (dThd, dUrd, and dCyd) while dGK phosphorylates purine-based dNs (dGuo and

dAdo). Human TK1 (hTK1) shares high amino acid sequence homology with those of

other vertebrates such as mouse, hamster, and chicken.6 The mouse dCK protein

sequence is 88% homologous to human dCK.7

Drosophila melanogaster (Fruit fly) has a single multisubstrate

deoxyribonucleoside kinase (Dm-dNK), which phosphorylates all four natural substrates

(dThd, dCyd, dGuo, and dAdo) with higher efficiency.8,9 Silk moth and mosquito also

contain a multisubstrate kinase, indicating that insects may only have a single type of

dNK.10,11

TK1-like sequences are found in several bacteria such as the Gram-positive

Bacillus anthracis and Staphylococcus aureus, the Gram-negative Escherichia coli, and

the mycoplasma bacterium Ureaplasma urealyticum.12-14 Lactobacilli and Bacilli possess

additional kinases to phosphorylate the other three natural substrates.15 However, E. coli

has only a TK activity but lacks dCK and dGK activities.14 On the other hand,

Mycobacterium tuberculosis is devoid of any TK activity.15

Several herpes viruses have TK activity including herpes simplex virus 1 and 2

(HSV1-TK and HSV2-TK), Varicella Zoster virus (VZV-TK), equine herpes virus 4

(EHV4-TK), Epstein-Barr virus (EBV-TK) and Kaposi's sarcoma-associated herpesvirus

4 (KSHV)/Human Herpesvirus 8 (HHV-8). Two major differences exist between hTK1 and viral TKs. First, all viral TKs except HSV2-TK have additional thymidylate kinase activity, which converts dTMP into dTDP. Secondly, viral TKs have a broad substrate specificity, and thus, phosphorylate purine and pyrimidine nucleosides while hTK1 accepts only endogenous dThd and dUrd. In particular, HSV-TK can convert therapeutic nucleosides with acyclic an sugar moiety, such as acyclovir, ganciclovir, and penciclovir, into the corresponding nucleoside monophosphates. Poxviruses are an exception among the viruses because it is believed that they contain a TK1-like kinase.16,17

HSV1- VZV- EBV- EHV hTK1 TK2 dCK dGK dNK UuTK CaTK TK TK TK 4-TK hTK1 n.s b n.s n.s n.s n.s n.s n.s 36% n.s 39 % (66674 a) TK2 34% 32% 48% n.s n.s 30% n.s n.s n.s (23304350) dCK 49% 31% n.s n.s 22% n.s n.s n.s (33357874) dGK 34% n.s n.s n.s n.s n.s n.s (17943105) Dm-dNK n.s 40% 22% n.s n.s n.s (31615910) HSV1-TK 31% 25% n.s 37% n.s (2624649) VZV-TK 25% n.s 39% n.s (33357776) EBV-TK n.s 29% n.s (9625651) UuTK n.s 47% (11357056) EHV4-TK n.s (38492740) CaTK

(15025935)

Table 1.1. Protein sequence identities of dNKs generated with BLAST 2.2.6 a Identification number of the proteins from the National Center for Biological Information (NCBI) b n.s: No significant similarity

5 Based on the amino acid sequences and substrate specificities, the known dNKs

are classified into three subgroups: 1) TK1-like kinases, 2) TK2, dCK, dGK, and Dm- dNK, and 3) viral TKs. As shown in Table 1.1, hTK1 has 36% and 39% sequence identities with Ureaplasma urealyticum thymidine kinase (UuTK) and Clostridium

Acetobutylicum thymidine kinase (CaTK), respectively. In the second group, there is at least 30% sequence identity among TKs while the sequence identities among the viral

TKs range from 25% to 39%. It should be noted that some scientists only postulate two subgroups of dNKs, the TK1-like kinases and the other dNKs.18,19

1.4. Crystal structures of dNKs

Several dNK X-ray crystal structures have been published. Table 1.2 summarizes

structural information (e.g., PDB ID, resolution, and ligands of dNKs deposited in

Protein Data Bank (PDB). Figure 1.3 shows three-dimensional (3D) X-ray crystal

structures of dNKs. X-ray crystal structures of human dNKs except TK2 were determined

during the last five years.20-24 The first X-ray crystal structure of a human dNK, the

mitochondrial dGK, was reported by Eklund and coworkers in 2001.21 Surprisingly, ATP

was found to bind into the substrate-binding site, not in the phosphate-donor site of the

dimeric 3D structure of dGK. Lavie and coworkers reported the crystal structure of dCK

in complex with its endogenous substrate dCyd and with the anticancer prodrugs AraC

and gemcitabine. dCK was also crystallized as a homodimeric protein.22 All dCK crystal

structures contained adenosine diphosphate (ADP) in the phosphate-donor site. Very

recently, dCK complexed with ADP and clofarabine was reported.24 Clofarabine is a

purine-nucleoside chemotherapeutic agent for the treatment of leukemias and other

6 hematological malignancies. The structural analysis of dCK-clofarabine-ADP complex

revealed that the purine-derived nucleoside (clofarabine) displays the same binding

pattern as the pyrimidine-based nucleosides (AraC and gemcitabine).24 Two different

groups reported crystal structures of hTK1 in complex with dTTP, which is the

endogeneous feedback inhibitor of this kinase.20,23 Both structures were crystallized as truncated tetramers, in which each monomer is missing 41 amino acids at the C-terminal region. It was reported, however, that removal of this C-terminal region did not affect

hTK1 activity.25

Dm-dNK structures were determined as dimers in complex with either of the two

substrates dThd or dCyd, or the feedback inhibitor dTTP.21,26 The substrate binding site

of Dm-dNK was similar to those of dGK and dCK. However, there was a significant

density for Mg2+ in the vicinity of β- and γ-phosphates binding sites in the Dm-dNK structure.26 The Mg2+ appeared to play a crucial role in inducing conformational changes

of Dm-dNK upon dTTP binding.26 Thymidine kinase from Ureaplasma urealyticum

(UuTK), a human pathogen in the urogenital tract that can cause pregnancy

complications, was solved as a tetramer in complex with dTTP or dThd.12,23 dThd alone

binds to the active site of UuTK in a similar way as the dThd portion in dTTP.12

Thymidine kinase from Clostridium acetobutylicum (CaTK), which is a Gram-positive, sporulating, anaerobe capable of naturally producing acetone, butanol, and ethanol, was co-crystallized with ADP. Due to the absence of substrate or feedback inhibitor, the substrate binding site appears to be more open compared to the hTK1 and UuTK.

dNKs PDB ID Resolution (Å) Ligands Reference 1XBT 2.40 dTTP 23 hTK1 1W4R 1.83 dTTP 20 1P60 1.96 dCyd and ADP 22 1P62 1.90 Gemcitabine and ADP 22 dCK 1P5Z 1.60 AraC and ADP 22 2A7Q 2.55 Clofarabine and ADP 24 2A2Z 3.02 dCyd and UDP 27 dGK 1JAG 2.80 ATP 21 2B8T 2.00 dThd 12 UuTK 1XMR 2.50 dTTP 23 CaTK 1XX6 2.00 ADP 1J90 2.56 dCyd 21 Dm-dNK 1OT3 2.50 dThd 26 1OE0 2.40 dTTP 26 1VTK 2.75 ADP and dTMP 28 2VTK 2.80 ADP and dThd 28 3VTK 3.00 ADP and 5-IdUMP 28 HSV1- 1KI2 2.20 Ganciclovir 29 TK 1KI3 2.37 Penciclovir 29 1KI7 2.20 5-iododeoxyuridine 29 1KI8 2.20 BVDU 29 1E2I 1.90 Acyclovir 30 VZV-TK 1OSN 3.20 BVDU-MP and ADP 31 1P6X 2.00 dThd 32 EHV4- 1P75 3.02 TP5A 32 TK 1P73 2.70 TP4A 32 1P72 2.10 dThd and ADP 32

Table 1.2. Structural information of dNKs doposited in Protein Data Bank

There have been several reports on the crystal structures of herpes simplex virus 1 thymidine kinase (HSV1-TK).28-30,33-35 X-ray crystal structures of HSV1-TK were determined in complex with various substrates including dThd, dTMP, 5-iodo-2´- deoxyuridine (5-IdUrd), 5-iodo-2´-deoxyuridine monophosphate (5-IdUMP), and the

8 antiviral drugs (E)-5-(2-bromovinyl)-2´-deoxyuridine (BVDU), acyclovir, ganciclovir,

and penciclovir.28-30,33-35 Thymidine kinase of varicella zoster virus (VZV-TK), which

causes both a chicken pox in children and shingles in adults, was determined as a monomer in complex with BVDU-MP and ADP.31 Thymidine kinase of equine herpes virus 4 (EHV4-TK), which causes respiratory disease and, less commonly, abortion in horses, was crystallized in complex with dThd and two so-called bisubstrate inhibitors with either four or five phosphate units covalently inserted between the 5´-postions of dThd and adenosine.32

1.5. Clinical implications of dNKs

The therapeutic activity of anticancer and antiviral agents derived from

nucleosides depends on the action of dNKs, which usually catalyzes the initial step in the

conversion of nontoxic nucleoside prodrugs into the corresponding toxic nucleoside

triphosphates. The anti-HIV prodrugs zidovudine (AZT) and stavudine (d4T) are activated

by hTK1,36,37 while anticancer prodrugs gemcitabine, cytarabine, clofarabine, cladribine, and fludarabine undergo monophosphorylation by the action of dCK.38-42 With the

exception of AZT, the initial 5´-monophosphorylation by hTK1 and dCK is the rate-

limiting step in this activation process.36-43 The antiviral agents acyclovir and ganciclovir

are converted into the monophosphates by HSV1-TK. Further phosphorylation is then carried

out by both viral and celluar enzymes in the host. The specific toxic effects towards lytic

viruses or proliferative cancer cells are predominantly exerted by the active triphosphate

forms of these prodrugs, which block DNA synthesis by inhibiting DNA polymerases or

HIV reverse transcriptase and by incorporating into 3´-terminal ends of DNA chains. 36-43

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

Figure 1.3. Crystal structures of dNKs

TK1-like family: (a) hTK1 with dTTP, (b) UuTK with dTTP, (c) CaTK with ADP. dCK, dGK, and Dm- dNK family: (d) dCK with dCyd and ADP, (e) dGK with ATP, (f) Dm-dNK with dCyd. Viral TK family: (g) HSV1-TK with dThd and ADP, (h) VZV-TK with BVDU-MP and ADP, (i) EHV4-TK with dThd and ADP

10 Furthermore, dNKs have become attractive candidates in suicide gene therapy and

cancer diagnostics. In suicide gene therapy, a dNK-encoded gene is inserted into the genome of target cells. The expressed dNK protein activates a specific nucleoside

prodrug, which is usually not activated by cellular dNKs in the host. The current most widely used combination is HSV-tk gene and ganciclovir.44-48 Tumors cells transfected

with the HSV-tk gene express the corresponding HSV-TK, which phosphorylates

ganciclovir selectively in the transfected cells. The monophosphate is further di- and

triphosphorylated by NMPK (and partially also HSV-TK) and NDPK, respectively.

Incorporation of ganciclovir triphosphates into the DNA chain leads to the termination of

DNA replication, and thus, resulting in tumor cell death.

More recently, dNKs have also been explored as molecular targets to detect tumor

progression and viral infections. Nucleoside prodrugs used for this purpose include 3´-

fluoro-3´-deoxythymidine (FLT), 1-(2-fluoro-5-methyl- β-arabinofuranosyl)uracil

(FMAU), and 1-(2-fluoro-5-iodo-β-arabinofuranosyl)uracil (FIAU), which have the

potential to be used as Positron Emission Tomography (PET) agents in radiolabeled

form.49-54

1.6. Human thymidine kinase 1 (hTK1)

1.6.1. hTK1 and TK2

Although TK2 is an isoenzyme of hTK1, it has significantly different biochemical properties. The human TK1 gene is located on chromosome 17 and that of TK2 is on

chromosome 16.55,56 Both enzymes do not share significant homology in their amino acid

sequences as shown in Table 1.1. The expression of hTK1 is cell cycle-dependent,

whereas the TK2 activity is constant throughout the cell cycle. The monomeric molecular 11 weights of hTK1 and TK2 are 25.5 kDa and 29 kDa, respectively.57,58 Both hTK1 and

TK2 phosphorylate dThd and dUrd efficiently, but TK2 can also accept dCyd as a

substrate. The limited dimensions of the dThd-binding site in hTK1 crystal structure

reflect its narrow substrate specificity. Nevertheless, hTK1 can phosphorylate dThd

analogues with modifications at the N-3, C-5 position, and the 3´-position of the 2´-

deoxyribose.4,59-62 In particular, bulky substitutions at the N-3 position of dThd are

tolerated by hTK1.59,61,62 On the other hand, TK2 primarily tolerates modification at the

2´-ara position of dThd or dUrd and it is also more susceptible to bulkier modfication

patterns at C-5 than hTK1.60,63

No hTK1 gene polymorphisms or phenotypic alterations have been reported in

humans until now. However, inactivation of the cytosolic TK1 gene in mice, which

eliminates dTMP synthesis via the salvage pathway, had a profound effect on their health, even though the de novo pathway remained intact under these conditions.64 On the other

hand, mutations in TK2 resulted in devastating myopathy and depletion of muscular

mitochondrial DNA in infancy.65,66

1.6.2. Biochemical properties of hTK1

During the cell cycle, the activity of hTK1 remains very low in early G1 phase,

begins to increase at late G1/S-phase, and reaches its maximum during S-phase, in which

DNA is synthesized. hTK1 is degraded during mitosis, and thus, undetectable during G1

phase. hTK1 activity is regulated during the cell cycle through phosphorylation.

Hyperphosphorylated hTK1 decreases affinity for dThd.67,68 Serine 13 of hTK1 is the

residue involved in the mitotic phosphorylation. Ser 13 is a highly conserved amino acid

12 residue in vertebrate TK1s. Mutation of serine 13 into alanine 13 resulted in abolishment

of mitotic phosphorylation of hTK1.67,68

ATP is a co-substrate of hTK1 and the phosphate donor for dThd and dUrd. It also acts as a positive effector, controlling a slow transition from low dThd-affinity hTK1

dimer (KM = 15 µM) to a high affinity tetramer (KM = 0.7 µM). hTK1 has a 20-fold

higher affinity for dThd in the presence of ATP than in the absence of ATP.69 The hTK1

dimer appeared to be dominant in G0 and G1 phases while the tetramer was

predominantly found in S phase.68

1.7. TK1 and drug design

TK2, dCK, and dGK are synthesized at relatively constant rates throughout the cell

cycle. hTK1 activity, however, is only found in proliferating cells, and thus, this enzyme is

widely distributed and expressed in malignant tumors,1,43 including those of the brain,70,71 pancreas,52 and lung.72 This makes the enzyme a very attractive molecular target for the

design of novel anticancer agents and tumor-prognostic markers. The recently determined

hTK1 crystal structures20,23 also allow structure-based drug design in silico. The work

described in this dissertation utilized hTK1 as a molecular target for the discovery of novel agents for the treatment of brain tumors by boron neutron capture therapy (BNCT), which will be discussed in Chapters 2-4. Knowledge and experience accumulated in the

anticancer project were then applied to discovery of novel antibiotics targeting BaTK,

which is the family of TK1-like kinases, for the treatment of anthrax infections (Chapter

5).

13 CHAPTER 2

BORON NEUTRON CAPTURE THERAPY AS A MODALITY TO TREAT

GLIOBLASTOMA MULTIFORME

2.1. Glioblastoma multiforme (GBM)

Glioblastoma multiforme (GBM) is one of the most malignant and aggressive of

all tumors. Despite significant advances in the treatment of many other cancer types using

surgery, chemotherapy, and radiation therapy, the survival of patients diagnosed with

GBM has remained almost unchanged for decades.73 The GBM accounts for

approximately 50% of newly diagnosed primary brain tumors in USA and Europe,73 and the incidence is 4.3 cases per 100,000 people per year from 1999 to 2002 in USA

(http://www.cdc.gov). Current standard therapy for GBM consists of surgical resection followed by radiotherapy. However, the 5-year survival rate for GBM patients over 45 is

2% or less and the median survival is generally less than one year from the time of diagnosis.74,75 This failure to cure GBM patients with surgery is due to the inability of

completely eradicating microinvasive tumor cells within the brain, mainly within a 2 cm-

margin of the surgical resection cavity.76-80 The success of conventional chemotherapy in

the treatment of GBM is also limited, probably because many anticancer agents cannot

14 penetrate the blood-brain barrier (BBB) effectively. The most commonly used

chemotherapeutic drugs for GBM are temozolomide and BCNU (carmustine). Radiation

therapy also has limited success in the treatment of GBM due to the damage to normal

brain tissue and spinal cord.

In a recent randomized trial with 573 GBM patients from 85 centers, 84% percent

of whom had undergone prior debulking surgery, radiotherapy plus temozolomide, a

conventional DNA methylating chemotherapeutic agent, resulted in a median survival of

14.6 months compared with 12.1 months for radiotherapy alone.81 This 2.5 months-

increase is probably one of the most significant documented improvements in the

treatment of GBM during the last three decades but it also bears testimony to the grim

reality that this form of cancer remains to be almost completely incurable in contrast with

other forms of cancer.82

Therefore, there is a pressing obligation to develop a broad range of novel

innovative treatment concepts and strategies for GBM. Several of these innovative

modalities have shown promising results in preclinical evaluation and clinical trials.

These include X-radiation sensitization,83-90 photodynamic therapy,91,92 signal transduction inhibition,75,93-99 angiogenesis inhibition,75,93-99 radiation therapies including

BNCT,70,100-104 immunotherapy including vaccines,105 gene therapy,74,75 oncolytic

viruses,74,75 and stem cell therapy.75 BNCT may have the potential to improve the

outcome of GBM treatment because it can direct radiation damage selectively to tumor

15 cells by employing molecular-targeting strategies.

2.2. Boron neutron capture therapy (BNCT)

2.2.1. Principles

BNCT is a binary chemotherapeutic method for the treatment of cancers,100 which is based on the nuclear reaction between boron-10 isotope (10B) and low energy thermal

neutrons. Both components alone are harmless in humans. However, when 10B atom is irradiated with thermal or epithermal neutrons, an unstable 11B isotope is produced,

which subsequently undergoes fission to yield two high linear energy transfer (LET)

particles: a 4He nuclei and a 7Li nuclei (Figure 2.1). These particles have very short path

lengths (9 and 5 µm, respectively), which approximate the diameter of a cell (10-15 µm).

Provided that 10B can be accumulated selectively in cancer cells, subsequent neutron

irradiation can selectively kill these cells without damaging the surrounding normal cells

due to the locally-restricted lethal effect.106

Figure 2.1. Nuclear capture and fission reaction by 10B and thermal neutron

2.2.2. Keys to success of BNCT

16 For successful BNCT, 10B-delivery agents should fulfill four criteria.100 First, a minimum accumulation of 20 µg of non-radioactive 10B per gram of tumor tissue, which

corresponds to ~ 109 boron atoms per tumor cell, is required. Secondly, 10B should be

delivered selectively to tumor cells and attained while at the same time the concentration

of 10B in surrounding normal tissue should be kept low to minimize damage to normal tissue. It is crucial that the ratio of tumor to brain (T/Br) and tumor to blood (T/Bl) boron

concentration is higher than 4. Thirdly, 10B should be retained in the tumor tissue for a

sufficient period of time to carry out BNCT. Finally, 10B-delivery agents should have

minimal systemic toxicity. Currently, no single boron-delivery agent satisfies all four

criteria.

2.2.3. Boron-delivery agents in clinical trials

Only two boron-containing compounds have been evaluated extensively in

clinical trials. One is (L)-4-dihydroxyborylphenylalanine (BPA), an amino acid

derivative,107-110 the other is sodium mercaptoundecahydro closo-dodecaborate (BSH), a

polyhedral borane anion (Figure 2.2).110,111 BPA has been used in the form of a fructose

complex because of its low water solubility.112,113 The BPA-fructose complex showed a

moderately selective accumulation in tumors cells. The advantages of BSH as boron- delivery agents are high boron percentage and low systemic toxicity. However, BSH was found to be very sensitive to oxidation when exposed to air.114,115 In addition, the T/Br

ratio of BSH ranged from 1.0 to 1.3. Although the safety of both drugs has been well

established, they have not demonstrated tumor-selective targeting. Therefore, there is an

urgent need to discover novel tumor-selective boron-delivery agents for BNCT.

2 HO SH B HO COOH B-H H2N B

BPA BSH

Figure 2.2. BNCT agents in clinical trials.

2.3. Boronated thymidine analogues as BNCT agents

2.3.1. Boron-containing cellular building blocks

Many BNCT agents have been designed and synthesized as boronated analogues

of endogenous cellular building blocks because tumors cells grow rapidly, and thus, have

an increased requirement for cellular building blocks compared with normal cells.106

Cellular building blocks used for conjugation with a boron moiety were e.g. nucleosides,59,62,116-120 amino acids,121-125 carbohydrates,126-128 polyamines,129-133 and

porphyrins.134-139 Prerequisite for the success of this strategy is, of course, that such

conjugates of biomolecules with the boron moiety are recognized by tumor cells in the

same way as the corresponding unaltered biomolecules.

Tumor cells require a substantial amount of nucleic acid precursors for DNA

biosynthesis. As discussed in Chapter 1, tumors cells synthesize dNTPs via salvage and de novo pathways. Boron-containing nucleosides, in particular derivatives of dThd and dUrd, have been intensively studied as BNCT agents because of their potential metabolic incorporation into tumors cells. 59,62,116-120 The biochemical pathway of these agents will be discussed in detail in Chapter 2.3.2. 18 Amino acids and related peptides are also required as cellular building blocks in

proliferating cells to a greater extent. BPA is known to accumulate selectively in tumor

cells based on its ability to serve as a substrate of the (L)-amino acid membrane

transporter,140-142 which is overexpressed in several forms of cancer due to the higher

demand for amino acids.143 Increased expression of the glucose transporter was also

found in tumor cells,144 and thus, boronated glucose could be taken up in tumors by

utilizing this elevated glucose transport system.

Polyamines (e.g. putrescine, spermidine, and spermine) are important biochemical

components for tumor cell proliferation and differentiation. Polyamine transport systems

are up-regulated in rapidly growing tumor cells, and thus boron-containing polyamines

could be transported more effectively into tumor cells.145 Boron-containing porphyrins also showed selective uptake and retention in tumor cells. It appears that these structures are “hitchhiking” with low-density lipoproteins (LDLs), which are selectively taken up by many tumor cells because they have an increased demand for cholesterol, and thus, overexpress LDL receptors on their cell membrane.134-139

2.3.2. Biochemical metabolic pathway of boron-containing thymidine analogues

A possible salvage pathway for activation of boron-containing dThd or dUrd

analogues is shown in Figure 2.3. Boronated dThd analogues (dThds) may enter cells

either via passive diffusion or facilitated and/or concentrative nucleoside transport.

Provided boronated dThds are good substrates for phosphorylating enzymes such as

hTK1, TMPK, or NDPK, metabolites of boronated dThds are accumulated in the cells due to the acquired phosphate groups. Nucleoside transporters do not accept nucleotides

19 as substrates and the negatively charged phosphate moieties prevent their own efflux by

passive diffusion. However, monophosphates (MPs) of boronated dThds could be

exported by nucleotide efflux pumps such as MRP-4 and MRP-5.146 The key regulatory

enzyme in the metabolism of boronated dThds presumably is hTK1. As discussed in

Chapter 1, the expression of this kinase is tightly cell cycle-regulated and active enzyme is found only in S-phase.18,147 Therefore, conversion of boronated dThds to the corresponding dTMPs by hTK1 may result in their selective transient intracellular

entrapment.

Figure 2.3. Biochemical salvage pathway of boron-containing thymidines.

20 It has been suggested that boron-containing nucleosides and nucleotides require access to cell nuclei in order to be active as BNCT agents148 and the hypothetical incorporation of boronated dNTPs into DNA has spurred early efforts to synthesize boron- containing nucleoside prodrugs as BNCT agents.106,149 However, in antimetabolite chemotherapy, active triphosphates are also responsible for most of the severe toxic side- effects, mainly in bone-marrow and intestinal epithelium.38-42 Triphosphates of boron- containing nucleoside prodrugs could exert a similar toxicity profile without neutron radiation when administered systemically at high doses. In particular, triphosphates of dThds substituted with a boron moiety at the N-3 position potentially terminate DNA synthesis because substitution at the N-3 position of thymine interferes with the base-pairing with the complementary adenine. Therefore, the monophosphorylation and/or diphosphorylation of boronated dThds by hTK1 and TMPK, and the subsequent transient intracelluar entrapment may be sufficient for BNCT.

2.3.3. hTK1 and brain tumors

TK1 and dCK activities of the rat F98 glioma, murine L929 TK1 (+), and L929

TK1 (-) cell lines were reported by Barth and coworkers.70 The rat F98 glioma cell line exhibits similar growth and invasive characteristics to human GBM,150 and therefore, it is considered as a suitable rat model for human GBM studies. As shown in Table 2.1, TK1 activities were 356 pmol/mg/minutes for F98 glioma and 228 pmol/mg/minutes for L929

TK1 (wt) cell lines, which express TK1. TK1 activities in F98 glioma and L929 TK1 (wt) cell lines were significantly higher than L929 TK1 (-) cells and normal brain. dCK activity was 3-5 fold higher in tumor cell lines than in normal brain. These data indicate

21 that hTK1 is an attractive target enzyme in the discovery of novel boron-delivery agents

for BNCT. It should be noted that murine TK1 and hTK1 have very similar substrate

specificities.151

Activity (pmol/mg/min)a Cell line TK1 dCK F98 glioma 356 ± 18 43 ± 5 L929 TK1 (wt) 228 ± 16 30 ± 2 L929 TK1 (-) 9 ± 3 29 ± 3 Normal brain (cortex) ≤ 5 ≤ 10

Table 2.1. TK1 and dCK activities in brain tumor cell lines and normal brain.70

a Activities of TK1 and dCK were determined by measureing the phosphorylation of [methyl-3H] dThd and [methyl-3H] dCyd using total protein extracts from the cell lines indicated.

2.3.4. Evaluation of hTK1 substrate characteristiscs of boron-containing dThds in

phosphorylation transfer assay (PTA).

Figure 2.4 shows the phosphorylation of dThd by hTK1, which catalyzes the

transfer of γ-phosphate group of ATP to the 5´-hydroxyl group of dThd or boron- containing dThds. The reaction by hTK1 produces dTMP and ADP. In a phosphoryl transfer assay (PTA), the [γ-33P]ATP is used as a phosphate donor. In the case of boronated dThds which are good substrate of hTK1, radioactive 33P is transferred from

[γ-33P]ATP to the 5´-hydroxyl group of boronated dThds to produce boronated

[33P]dTMPs. Phosphorimaging is utilized to quantitate the production of radioactive

boronated dTMPs.

Figure 2.4. 5´-Monophosphorylation of dThd or boronated dThds by hTK1

2.4. Carboranes as boron moieties of BNCT agents

The early interest in the polyhedral boron and carborane cluster was their

development and use as high-energy fuels for aircrafts and rockets in 1950-60s.152,153

Another interest that emerged soon thereafter was their medical application in BNCT. In particular, carboranes (C2B10H12) are advantageous for drug design in BNCT because of

their high boron content and stability.106 Synthetic and structural versatility as well as lack of toxicity are other beneficial factors.154

2.4.1 Carborane structures

Polyhedral carboranes (C2B10H12) are three-dimensional σ-aromatic structures.

They are composed of two carbon atoms, ten boron atoms, and twelve hydrogen atoms.

One of the striking structural features of carboranes is their icosahedral geometry in 23 which the carbon and boron atoms are hexavalent. Closo-carboranes are available in the form of three geometrical isomers (ortho, meta, and para) based on the position of the two carbon atoms in the cage scaffold (Figure 2.5).

closo-o-Carborane 1 nido-o-Carborane H 5 2 - [c-o-C2B10H12] [n-o-C2B9H12 ] 9 7

12 nido-m-Carborane = BH closo-m-Carborane 1 H - = CH [c-m-C2B10H12] [n-m-C2B9H12 ] 7 closo-p-Carborane nido-p-Carborane 1 H [c-p-C B H ] - 2 10 12 [n-p-C2B9H12 ]

Figure 2.5. Closo- and nido structures of o-, m-, and p-carborane

2.4.2. Preparation of carboranes

Closo o-carborane can be prepared in two steps by refluxing decaborane (B10H14)

in acetonitrile, followed by refluxing the resulting decaborane-acetonitrile complex with acetylene in toluene or benzene.154,155 Recently, reaction of decaborane with alkynes in the presence of ionic liquids produced closo o-carboranes in high yields.156 Closo m-

carborane is produced by thermal rearrangement of closo o-carborane at 400-500 °C.

Closo p-carborane is obtained via the same process by exposing closo m-carborane to

600-700 °C. Since the preparation of o-carboranes is the simplest of the three isomers, it

is the least expensive, and therefore, has been studied extensively both in naturally-

ocurring abundance and 10B-enriched form. 24 Cage degradation of neutral closo o-carborane into negatively charged water-

soluble nido o-carborane occurs by treatment with Brønsted bases such as amines,

alkoxides and hydroxides.154 Although m-carborane is more resistant to the degradation

by Brønsted bases than o-carborane, it is easily converted into water-soluble nido m-

carborane by treatment with the fluoride ion.157-160 Transformation of closo p-carborane

into nido p-carborane requires extremely harsh reaction conditions.161

2.4.3. The chemistry of carboranes

Carboranes are in general extremely stable to oxidative, reductive, and acidic

conditions. However, substitution reactions at both C-H and B-H vertices of these

clusters occur under specific conditions. The hydrogen atoms attached to the carbon

atoms can be removed by the treatment of n-butyllithium (n-BuLi) in benzene,

tetrahydrofuran (THF), or diethylether. Reported pKa values of o-, m-, and p-carborane in

ethanol are 24, 33 and 40, respectively.162 As a result, C-H vertices of o-carborane are

activated more easily than those of m- and p-carborane The weakly acidic C-H protons in carboranes have the potential for hydrogen bond formation162,163 and their overall

hydrophilicity decreases in the order o- > m- > p-carborane.164 C-lithium salts of all three

carborane isomers undergo nucleophilic substitution reactions with alkyl halides or tosylates.

Since carboranes are aromatic, they also undergo electrophilic substitution in a fashion similar to benzene. The B-H vertices antipodal to the carbon atoms of closo o- carborane (9B and 12B of closo o-carborane in Figure 2.5) are more susceptible to electrophilic substitution than the other B-H vertices due to the electronegativity

25 difference between carbon and boron.154 Halogen atoms can be introduced preferentially

at both antipodal boron atoms using molecular halogens in the presence of Lewis acids

such as aluminum chloride.165,166 They can be replaced with alkyl, allyl, aryl, or other

halides using palladium-catalyzed cross-coupling reactions.167-169

2.4.4. Theoretical calculations of carboranes

Structural determinations of carboranes have been achieved by gas-phase electron

diffraction.170,171 However, there are no reported X-ray crystal structures of carboranes

due to the cage disorder by rotation of the psedo-spherical cluster and difficulties in

distinguishing between the boron and carbon atoms. By utilizing the C-H acidity of

carboranes, crystal structures of carboranes have been determined in complex with

molecules containing heteroatoms (e.g, N, O, S, P, and halogens), which are able to

participate in hydrogen-bonding with the acidic C-H of carboranes. The structural data

obtained experimentally have been utilized to construct more accurate carborane models

theoretically. Semi-empirical, ab initio Hartree-Fock (HF), and density functional theory

(DFT) calculations of all carboranes have been carried out by a number of research

groups.171-174 Geometries and parameters (e.g. bond length, bond angle, and charge) of

carboranes calculated at the HF and DFT levels were in good agreement with those determined experimentally.171-174 However, structural information of carboranes obtained

from theoretical calculations have not been utilized for docking studies of carborane-

containing compounds with macromolecules because current molecular docking

programs (e.g. Sybyl, Autodock) do not provide parameters for the hexavalent boron

atom of carboranes.

2.5. Carborane-containing dThds.

2.5.1. dThds substituted with a carborane cage at the carbohydrate portion

The first reported carborane-containing nucleoside was a uridine derivative

substituted with carborane at the 2´-position.175 A number of additional uracil/thymine-

based nucleosides with carborane cages linked to the 2´- , 3´-, or 5´-positions of ribose or

2´-deoxyribose were synthesized.119,120,176 PTAs with recombinant hTK1 showed that

introduction of the carborane cluster at the 3´-position of dThd is moderately tolerated by

hTK1.120

2.5.2. dThds substituted with a carborane cage at the C-5 position

A basic motivation of the strategy to introduce the carborane cluster at the 5-

position of dUrd or dThd certainly stemmed from the fact that substitution of CH3 at the

C-5 position of dThd with iodine and bromine resulted in dUrd prodrugs such as 5-iodo- and 5-bromo-2´-deoxyuridine, which are excellent substrates for phosphorylating enzymes. Indeed, metabolites of both prodrugs are able to substitute for dTTP during

DNA synthesis to a high extent.177 Among dThds and dUrds substituted with the

carborane cage at the 5-postion, CDU [5-(1-o-carboranyl)-2´-deoxyuridine, Figure 2.6] was selected as a potential candidate for BNCT and further evaluated in vitro and in vivo.118,178

27 H O O N OH HN HO N O OH HO O N O O OH OH CDU N5-2OH

Figure 2.6. Potential dThd-based BNCT agents in preclinical trials

2.5.3. dThds substituted with a carborane cage at the N-3 position (3CTAs)

In 1996, Lunato reported that dThds substituted with a carborane cluster at the N-

3 position of dThd were phosphorylated by recombinant hTK1.179 These novel dThd- based prodrugs with a carborane cage at the N-3 position were later termed 3-carboranyl thymidine analogues (3CTAs). Since the first report of 3CTAs appeared,179 3CTAs have

been extensively studied as boron-delivery agents for BNCT by Tjarks, Eriksson, Barth,

and their coworkers.59,61,62,116 A series of 3CTAs includes dThds substituted with

hydrophilicity-enhancing dihydroxypropyl group at the carborane cage or flexible

ethyleneoxide moieties between the dThd scaffold and carborane cage.59,61 One of these

3CTAs, designated N5-2OH (3-[5-{2-(2,3-dihydroxyprop-1-yl)-o-carboran-1-yl}pentan-

1-yl]thymidine, Figure 2.6), was identified as a lead compound and extensively evaluated

biochemically and biologically. N5-2OH had a good hTK1 phosphorylation rate (41%

relative to that of dThd) and the highest kcat/KM value of all 3CTAs (35.8% relative to that of

dThd).70 Furthermore, in the absence of endogenous dThd, N5-2OH was a better substrate of

hTK1 than FLT (kcat/KM = 7.6% that of dThd) and comparable to AZT (kcat/KM = 43.7% that

of dThd).180 In the presence of dThd, N5-2OH was a moderate inhibitor of dThd

28 181 phosphorylation with an IC50 of 9.3 µmol/L. However, N5-2OH was not phosphorylated by other dNKs including TK2, Dm-dNK, and dCK.182

29 CHAPTER 3

10B-ENRICHED N5-2OH:

A PROMISING BORON-DELIVERY AGENT FOR BNCT

3.1. Background and rationale

N5-2OH was selected as the first lead compound of our 3CTA library because it

exhibited higher phosphorylation rates (PRs) by hTK1 and superior in vitro biological

properties compared with other 3CTAs.59,70,181 It also showed physiochemical properties

suitable for effective penetration of the BBB (logP = 2.09).181 Some biological data of

N5-2OH are summarized in Table 3.1.

Although N5-2OH was found to be a promising boron-delivery agent for BNCT of GBM, some drawbacks were identified as well. First, preparation of a sufficient amount of N5-2OH in 10B-enriched form was a prerequisite to investigate its activity in neutron irradiation experiments with tumor/rodent models. The isotopes of the element boron occur with a natural abundance of 19.8% for 10B and 80.2% for 11B. However, only

10B has the capacity to capture thermal neutrons effectively, and thus, can be utilized for

BNCT.106 In order to reduce their quantities by a factor of 4, and consequently, decrease systemic toxicity, clinical BNCT agents are usually administered in 10B-enriched form.106

30 Both clinical BNCT agents, BPA and BSH discussed in Chapter 2, have been synthesized

and evaluated in 10B-enriched form.100,148

Experiment Model Value hTK1 phosphorylation 41 ± 5a Phosphorylationa TK2 phosphorylation 0.002b Rat F98 glioma 70 µM In vitro Murine L929 fibroblast (wt) 23 µM cytotoxicityc Murine L929 TK1 (-) 58 µM Rat F98 cell line 25.7 ± 6.0 µg B/109 cells In vitro Murine L929 (wt) 92.0 ± 20.0 µg B/109 cells uptaked Murine L929 TK1 (-) 9.3 ± 2.7 µg B/109 cells

Table 3.1. Summary of biological data of N5-2OH70

a Value was determined using 10 µM of N5-2OH as described in Chapter 2.3.4 and expressed relative to dThd. The value of dThd was set to 100. b Value was determined as described in Chapter 2.3.4 using 100 µM of N5-2OH c The values represent the concentration of N5-2OH, which produced a 50% reduction in cell viability. dCells were incubated with 17.5 µM of N5-2OH for 24 h at 37 °C, followed by harvest and boron determination by means of DCP-AES.183

Secondly, the evaluation of optically pure epimers and geometrical isomers of

N5-2OH was crucial to complete structure-activity relationship (SAR) studies. Previous

biological studies were carried out with the mixture of two epimers of the o-carboranyl

form of N5-2OH, which have (R)- and (S)-configuration at C-16 marked with ‘*’ in

Scheme 3.1. Therefore, both stereochemically pure forms of o-carboranyl N5-2OH as

well as geometrical isomers derived from m- and p-carborane were prepared.

31 Finally, the elucidation of the binding mode of N5-2OH in the active site of hTK1

was not possible until the end of 2004, when the first hTK1 crystal structure was

published.23 hTK1 has a very narrow substrate specificity compared with the other dNKs, accepts only two natural substrates (dThd and dUrd) and only minor modifications at both nucleosides are tolerated.1,18 For the design of improved 3CTAs, it was of pivotal

importance to understand why N5-2OH and other 3CTAs, having a bulky carborane

cluster and a long alkyl or ethylene oxide spacer group at the N-3 position,59,61,62 were accepted as substrates of hTK1. Therefore, detailed computational docking studies with various geometric N5-2OH isomers using both hTK1 crystal structures and a homology

model of hTK1 were carried out.

3.2. Preparation and analysis of 10B-enriched N5-2OH

3.2.1. Synthesis

The original 10-step synthesis of o-carboranyl N5-2OH with naturally-occurring

boron isotope distribution (termed as 11B-abundant) and as a mixture of two epimers was

reported by Al-Madhoun and coworkers.59 Although this lengthy procedure was suitable

for the preparation of sufficient quantities of N5-2OH and several of its homologues for

initial SAR studies, it was found to be inefficient and costly for the synthesis of N5-2OH

in 10B-enriched form.

To obtain 10B-enriched N5-2OH, two possible synthetic routes were designed

utilizing commercially available 10B-enriched o-carborane or decaborane. The first 4-step

route, starting from 10B-enriched o-carborane (98% 10B), is shown in Scheme 3.2.

Scheme 3.3 shows an alternative synthetic sequence starting from 10B-enriched

32 decaborane (99.5% 10B). Prior to the use of expensive 10B-enriched starting materials,

11B-abundant N5-2OH was synthesized to identify optimal reaction conditions.

a HO b AcO c HO H H

AcO H AcO * AcO OH d e OH

AcO f O g HO O h O O

N O TsO O i HO N O O j O O

O ∗ N OH HO N O OH O

OH N5-2OH

Scheme 3.1. Synthesis of 11B-abundant N5-2OH by Al-Madhoun et al.59

Reagents: (a) 3-aminopropylamide, 1,3-diaminopropane; (b) AcCl, pyridine, Et2O; (c) B10H14, CH3CN, toluene; (d) Tris(dibenzylideneacetone)dipalladium, bis(diphenylphosphino)ethane, allylethyl carbonate, THF; (e) OsO4, NMO, THF; (f) p-TsOH, CH3C(OCH3)2CH3; (g) K2CO3, acetone, H2O; (h) p-TsCl, Et3N, DMAP, CH2Cl2, (i) dThd, K2CO3, DMF, acetone; (j) 17% HCl, CH3OH.

33 H H H TsO a b c

o-carborane 3.2.A 3.3.A 3.2.B 3.3.B

O O N d N OH HO OH HO N O N O O O

OH OH 3.4.A 3.5.A (11B-abundant N5-2OH) 3.4.B 3.5.B (10B-enriched N5-2OH)

Scheme 3.2. Preparation of 10B-enriched N5-2OH from o-carborane

Reagents and reaction conditions: (a) n-BuLi, allyl bromide, THF, rt, 12 h; (b) n-BuLi, 1,5-pentanediol di- o o p-tosylate, benzene, 5-10 C, 1 h; (c) dThd, K2CO3, DMF/acetone (1:1), 35 C, 96 h; (d) OsO4, NMO, 1,4- dioxane.

Reaction of an o-carborane lithium salt, generated from o-carborane by treatment with n-BuLi in THF at -78 °C, with allyl bromide provided compound 3.2.A.

Homobifuntional 1,5-pentanediol di-p-tosylate was chosen to connect the carborane cage with the N-3 position of dThd through a pentylene spacer. Treatment of 1,5-pentanediol with p-toluenesulfonyl chloride and triethylamine in dichloromethane, followed by recrystallization in ethyl acetate, afforded 1,5-pentanediol di-p-tosylate as a white solid in

75% yield. To prevent formation of by-products 3.3.C and 3.3.D (Figure 3.1) during the second step of Scheme 3.1, the lithium salt of 3.2.A was dissolved in benzene and the resulting solution was added very slowly to a solution of 1,5-pentanediol di-p-tosylate in benzene at 5-10 °C. Stirring of the reaction mixture at 5-10 °C for 1 h and subsequent quenching with water afforded compound 3.3.A in 70% yield.

34 a o-carborane 3.2.A

3.2.C H

b 3.2.A 3.3.A 3.3.C 3.3.D O N c HO N O 3.3.A 3.4.A O 3.4.C OH

Figure 3.1. By-products observed in steps a, b and c of Scheme 3.2.

Previously, it was reported that compound 3.2.A isomerizes in the presence of

potassium tert-butoxide in tert-butanol or benzene at 40-45 °C to produce 1-(trans-1-

propenyl)-o-carborane 3.3.D.184,185 The formation of 3.3.D was observed at room temperature. However, lowering the reaction temperature to 5-10 °C significantly reduced the formation of 3.3.D. Isomerization of the allyl group was also observed in step c of Scheme 3.2. When compound 3.3.A was reacted with dThd in DMF/acetone (1:1) at

35 °C for 96 h, 3.4.A was obtained in a ratio of > 30:1 with the olefin isomer 3.4.C,

which was not easily separated from 3.4.A by silica gel chromatography. Higher

temperature (50 °C) reaction condition produced 3.4.A and its olefin isomer 3.4.C in a

1:1 ratio. When the mixture of 3.4.A and 3.4.C was treated with the osmium tetroxide

(OsO4) and 4-methylmorpholine N-oxide (NMO), only 3.4.A was dihydroxylated to

35 afford target compound 3.5.A. Compound 3.4.C appeared to be more resistant to the dihydroxylation reaction presumably due to steric hindrance by the bulky o-carborane cluster. Using reaction conditions optimized for the preparation of 3.5.A, 10B-enriched

N5-2OH (3.5.B) was obtained in 4 steps from 10B-enriched o-carborane in 15% overall

yield by following the same synthetic route.

Si H Si a b c d-f B10H14 Si 3.2.B 3.5.B Decaborane 3.6 3.7

Scheme 3.3. Synthesis of 10B-enriched N5-2OH from decaborane

Reagents and reaction conditions: (a) (bmin)Cl, toluene, reflux, 10 min; (b) n-BuLi, allyl bromide, THF, rt, o 12 h; (c) TBAF, THF; (d) n-BuLi, 1,5-pentanediol di-p-tosylate, benzene, 5-10 C, 1 h; (e) dThd, K2CO3, o DMF/acetone (1:1), 35 C, 96 h; (f) OsO4, NMO, 1,4-dioxane.

The alternative synthetic route for 10B-enriched N5-2OH (3.5.B), starting from

10B-enriched decaborane, is shown in Scheme 3.3. By employing biphasic reaction conditions reported by Sneddon and coworkers,156 the reaction of decaborane with

trimethylsilyl acetylene in a mixture of 1-butyl-3-methylimidiazolium (bmin) chloride

and toluene afforded compound 3.6 in 50% yield. In contrast, the conventional reaction

of a decaborane-acetonitrile adduct with trimethylsilyl acetylene,154 gave compound 3.6

in only 23% yield. Lithium salt formation of the compound 3.6 with n-BuLi, followed by

addition of allyl bromide, provided compound 3.7 in 60% yield. Removal of the

trimethylsilyl group of 3.7 using tetrabutylammonium fluoride (TBAF)186 gave

36 compound 3.2.B (80% yield), which was further processed as described in Scheme 3.2, to

obtain target compound 3.5.B.

3.2.2 Analysis

Infrared (IR) and high-resolution mass spectroscopy (HRMS) analysis of 11B- abundant N5-2OH (3.5.A) and 10B-enriched N5-2OH (3.5.B) revealed interesting

differences as a result of the isotopic composition of both compounds. The IR spectra

exhibited minor differences in the B-H stretching band of 2580 cm-1 for 3.5.A versus

2587 cm-1 for 3.5.B. Electrospray ionization (ESI) mass spectra of 3.5.A and 3.5.B

showed significant differences in their isotope patterns. The base peak of 3.5.A (M + Na)

+ centered around 551 Da while that of 3.5.B (M + Na)+ appeared at 543 Da (Figure 3.2).

A gaussian-shaped curve distribution, which is typical for 11B-abundant carborane-

containing molecules, was only observed in HRMS spectrum of 3.5.A.

Figure 3.2. ESI-mass spectra of compounds 3.5.A and 3.5.B 37 Recently, it was reported that the reaction of dThd with 8-dioxane-3-cobalt bis(dicarbolide) using sodium hydride as a base produced a mixture of O-4 and N-3 alkylated product.176 Since there was also a possibility of producing O-4 alkylated product when dThd was condensed with the carboranyl alkyltosylate under the mild basic conditions (potassium carbonate in acetone/DMF at 35 °C) used in our studies, selective

N-3 alkylation was confirmed using 2D-NMR studies.

Figure 3.3. 2D-HMBC spectrum of compound 3.5.A

38 A previous analysis by 1D-NMR confirmed N-3 alkylation in N5-2OH solely by

13 179 typical differences of C-chemical shift between 4-OCH2- and 3-NCH2- groups. The

2D-HMBC (Heteronuclear Multiple Bond Correlation) NMR was selected because it

shows the coupling between proton nuclei and carbon nuclei, which are separated by one

or two bonds. Thus, if O-4 alkylated product had been generated under the mild basic

condition, the hydrogens at C-8 would have coupled with C-4 and C-5. However, they

showed coupling with C-2 and C-4, establishing that it is N-3 alkylated product. Prior to

the 2D-HMBC experiment, each proton and carbon peak of compound 3.5.A was

assigned by 1H-NMR, 1H-1H COSY, 13C-NMR, 13C-DEPT, and 1H-13C HMQC (all of the

spectra are shown in the Appendix A). As shown in Figure 3.3, two hydrogens at C-8

coupled with both carbonyl carbons (C-2 and C-4). Thus, the 2D-HMBC experiment

confirmed exclusive N-3 alkylation in compound 3.5.A.

3.3. Preparation and analysis of pure N5-2OH epimers and geometrical isomers

3.3.1. Synthesis

The synthetic procedure (Scheme 3.1) reported by Al-Madhoun et al.59 and the

synthetic routes described in Scheme 3.2 and 3.3 provided N5-2OH as a mixture of two

epimers due to dihydroxylation of the allylic function with OsO4. The epimers have

different configuration at the C-16 position of N5-2OH. Since the pure stereoisomers are

more potent and less toxic than a mixture in some cases of drug development, pure

epimers of N5-2OH were prepared from chiral starting materials as shown in Scheme 3.4.

Compound 3.8.A was prepared in 63% yield by reacting o-carborane with (S)-(+)-2,2- dimethyl-1,3-dioxolane-4-ylmethyl p-tosylate. Addition of a solution of the lithium salt

39 of compound 3.8.A in benzene to a solution of 1,5-pentanediol di-p-tosylate in benzene

gave compound 3.9.A in 58% yield.

H H H * TsO * O O a b c O O

3.8.A: (R) 3.9.A: (R) 3.8.B: (S) 3.9.B: (S) O O R * N N O d HO N O O HO N O O O

OH OH

OH 3.10.A: (R) 3.11.A: (R)-epimer R= 3.10.B: (S) OH 3.11.B: (S)-epimer R= OH OH

Scheme 3.4. Synthetic route for pure N5-2OH epimers

Reagents and reaction conditions: (a) n-BuLi, (S)- or (R)-2,2-dimethyl-1,3-dioxolane-4-yl methyl p-tosylate, o benzene, rt, 14 h; (b) n-BuLi, 1,5-pentanediol di-p-tosylate, benzene, 5-10 C, 1 h; (e) dThd, K2CO3, DMF/acetone (1:1), 50 oC, 48 h; (f) 17% HCl, MeOH.

Condensation of compound 3.9.A with dThd, followed by deprotection of the isopropylidene group under the acidic condition, afforded compound 3.11.A with (R)-

configuration at C-16. The second epimer (3.11.B) was prepared from (R)-2,2-dimethyl-

1,3-dioxolane-4-ylmethyl p-tosylate in accordance with the procedure described for

3.11.A. It should be noted that this synthetic route is potentially suitable for the

preparation of N5-2OH in 10B-enriched form. In addition, this synthetic route eliminates

40 the use of toxic OsO4, which was applied for the dihydroxylation of the allylic group in

the reaction sequences shown in Schemes 3.1, 3.2, and 3.3.

a X b TsO c Carboranes O X O O O

3.8.C: X= o-carborane 3.9.C: X= o-carborane 3.8.D: X= m-carborane 3.9.D: X= m-carborane 3.8.E: X= p-carborane 3.9.E: X= p-carborane O O OH N X N O d OH HO O N O HO N O O O 3.11.C OH OH 3.10.C: X= o-carborane 3.10.D: X= m-carborane O OH 3.10.E: X= p-carborane N OH HO N O O

OH 3.11.D

O N HO N O O OH OH 3.11.E OH

Scheme 3.5. Synthetic route for geometrical N5-2OH isomers

Reagents and reaction conditions: (a) n-BuLi, 2,2-dimethyl-1,3-dioxolane-4-yl methyl p-tosylate, benzene, o rt, 14 hr; (b) n-BuLi, 1,5-pentanediol di-p-tosylate, benzene, 5-10 C, 1 hr; (e) dThd, K2CO3, DMF/acetone (1:1), 50 oC, 48 h; (f) 17% HCl, MeOH.

The synthetic approach described in Scheme 3.4 was also used for the preparation of the m-, and p-carboranyl isomers of N5-2OH. (R/S)-mixtures of compounds 3.11.C,

41 3.11.D, and 3.11.E were synthesized in 4 steps from o-, m- and p-carborane in overall

15%, 19%, and 16% yield, respectively (Scheme 3.5).

3.3.2. Analysis

A chiral solvating agent was used to determine the ratio of (R)- and (S)-epimers of

3.5.A. When one equivalent of (S)-(+)-2,2,2-trifluoro-1-(9-anthryl)-ethanol (Pirkle

187 1 alcohol) was treated with 3.5.A in CDCl3, the H-NMR spectrum showed a partial

overlap of two triplets for H-1´ with different intensities (Figure 3.4). In contrast, when

the pure epimers 3.11.A and 3.11.B were treated with the Pirkle alcohol under the same

conditions, single triplets for H-1´ appeared at 6.13 and 6.10 ppm, respectively. Two

signals were also observed for the hydroxyl proton at C-16 of 3.5.A and 3.11.C while

only one single peak was observed for that of the pure epimers (3.11.A and 3.11.B).

Based on this analysis, compound 3.5.A consisted of approximately 3.11.A and 3.11.B in a 2:3 ratio. Steric hindrance by o-carborane and close proximity of thymidine to the allylic group appeared to cause the unequal compositions of (R)- and (S)-epimers of

6 3.5.A. When acetone-d was used instead of CDCl3, no splitting pattern or chemical shift

change was observed, probably because of the carbonyl group of acetone-d6, which prevented the OH group of Pirkle alcohol from forming hydrogen bonds with OH group

(C-16) of 3.5.A.

Figure 3.4. Partial 1H-NMR spectra of 3.11.A, 3.11.B, 3.11.C, and 3.5.A with (S)-(+)- 2,2,2-trifluoro-1-(9-anthryl)-ethanol in CDCl3 at 300 K

The optical rotation values for 3.5.A (-5.1), 3.11.A (+17.5), and 3.11.B (-14.8)

indicated a similar epimeric composition for 3.5.A. Efforts to determine the epimeric

ratio of 3.5.A by HPLC using either a reversed phase (C18) column or a β-cyclodextrin-

based chiral column (CYCLOBOND I, Advanced Separation Technologies Inc.) were not

successful. We did also not observe any differences in CD spectra of epimers 3.11.A and

3.11.B presumably due to the fact that C-16 of N5-2OH is separated from thymine, the major chromophore, by mulitple bonds.

Purity verification of all target compounds by analytical reversed-phase (C18)

HPLC using methanol/water and acetonitrile/water gradient systems revealed interesting

retention times, in particular for the geometrical isomers of N5-2OH (Table 3.2).

43 Water/acetonitrile gradient (100:0 to 70:30 over 5 min, from 70:30 to 40:60 over 25 min,

from 40:60 to 0:100 over 20 min) with a flow rate of 1 mL/min was applied to determine

retention times of 3.11.C, 3.11.D, and 3.11.E. As already discussed in Chapter 2.4.3, the

hydrophobicity of three carborane isomers decreases in the order of p- > m- > o-

carborane presumably due to the decreasing potential of C-H for hydrogen bond

formation.162,164 Using the water/acetonitrile gradient system, however, the retention times of the o- (3.11.C), m- (3.11.D), and p-(3.11.E) carboranyl N5-2OH isomers, and

thus their hydrophobicities decreased notably in the order of 3.11.C > 3.11.D > 3.11.E

(Table 3.2), which is also demonstrated by the HPLC chromatogram of a mixture of the three isomers shown in Figure 3.5.

Figure 3.5. HPLC spectrum of a mixture of geometrical N5-2OH isomers (3.11.C, 3.11.D, and 3.11.E) using water/acetonitrile gradient

44 This trend was also observed when methanol/water gradient was used.

Accordingly, the ratios of apolar surface areas (APSA) to polar surface areas (PSA) for

3.11.A, the (R)-epimer of 3.11.D, and the (R)-epimer of 3.11.E, calculated with VEGA

ZZ 2.0.4 software (Milano, Italy)188 decreased in the order 3.11.A > (R)-3.11.D > (R)-

3.11.E (Table 3.2).

Retention time Compound hTK1 PRa (APSA/PSA) ratioc (RP-18)b N5-2OHd 39 ± 8 26.9 min

3.5.A 39 ± 6 26.9 min

3.5.B (10B enriched) 36 ± 7 26.5 min

3.11.A (R) 34 ± 7 26.7 min 3.31 (461.4 Å2 / 139.3 Å2)

3.11.B (S) 33 ± 6 26.6 min

3.11.C (R/S, o-) 32 ± 7 26.9 min

3.11.D (R/S, m-) 42 ± 4 25.7 min 3.16 (492.7 Å2 / 156.1 Å2)

3.11.E (R/S, p-) 45 ± 6 23.7 min 3.02 (491.8 Å2 / 162.9 Å2)

Table 3.2. hTK1 PRs, retention time, and ratios of apolar to polar surface area (APSA/PSA) of the N5-2OH isomers

a Compound concentrations were 40 µM and the DMSO concentration was set to 1%. Enzyme activities were determined using a spectrophotometric assay. b Water/acetonitrile gradient (100:0 to 70:30 over 5 min, from 70:30 to 40:60 over 25 min, from 40:60 to 0:100 over 20 min) with a flow rate of 1 mL/min was applied. c PSAs and APSAs of compounds 3.5.A and (R)-epimers of 3.11.D and 3.11.E were calculated using the VEGA ZZ 2.0.4 program. d N5-2OH was synthesized using the method reported by Al-Madhoun et al.59

45 3.4. In silico Studies

Recently, crystal structures of hTK1 (PDB ID: 1W4R and 1XBT),20,23 UuTK

(PDB ID: 1XMR and 2B8T),12,23 and CaTK (PDB ID: 1XX6) were published and are now available for structure-based drug design. The crystal structures of hTK1 and UuTK

were determined as tetramers containing dThd or dTTP.20,23 However, CaTK was

crystallized as a dimer in complex with ADP. A visual inspection indicated that the

hTK1s and UuTKs may represent “closed” TK forms while CaTK may constitute an

“open” or “semi-open” TK form. In the case of many other nucleoside kinases and

nucleoside monophosphate kinase, binding of the substrates and ATP appears to be

associated with a conformational change from an open unoccupied form, over a partially

closed form involving substrate- or ATP binding, to a closed form binding both substrates

and ATP.189-193

The structures of 3.11.A and the (R)-epimers of 3.11.D and 3.11.E were

optimized at the level of B3LYP/6-31G* using the Gaussian 03 Program194 running on an

Itanium 2 Cluster at the Ohio Supercomputer Center (Figure 3.6). Conformational and

geometrical parameters of the dThd scaffold of all three compounds were compared with

the analyzed dThd structure reported by Ghomi and coworkers.195-197 The lowest energy

conformer of dThd has C-2´-endo/anti conformation.195-197 The glycosyl torsion angle (O-

4´, C-1´, N-1, and C-2) in dThd was found to be between 90° and 270° for the anti

orientation and between -90° and +90° for the syn orientation.195-197 Quantum mechanical

calculations of 3.11.A and the (R)-epimers of 3.11.D and 3.11.E at the B3LYP/6-31G* level showed a stable C-2´-endo/anti conformation of dThd scaffold in all three compounds. The glycosyl torsion angles of optimized 3.11.A and the (R)-epimers of

46 3.11.D and 3.11.E were +232°, +229°, and +229°, respectively. Also, all of them were C-

2´-endo conformers. Interestingly, the distance between O-3´ and the hydrogen of the OH

group attached to the C-17 position of 3.11.A was 2.005 Å, and thus, intramolecular

hydrogen bonding is conceivable. This specific interaction may render 3.11.A more

amphiphilic than the (R)-epimers of 3.11.D and 3.11.E (Figure 3.6), which may also explain the observed differences in retention times in HPLC experiments and APSA/PSA ratios (Table 3.2).

Figure 3.6. The optimized structures of 3.11.A and (R)-epimers of 3.11.D and 3.11.E at the B3LYP/6-31G* level using Gaussian 03 program.

Structures were visualized with GaussView3.0 (Gaussian Inc., Pittsburg, PA). Each solid sphere stands for atoms as follows: Red (oxygen), blue (nitrogen), dark gray (carbon), pink (boron), light gray (hydrogen)

For docking studies, the coordinates of 3.11.A and the (R)-epimers of 3.11.D and

3.11.E were transferred into Sybyl 7.1 (Tripos Inc., St. Louis, Missouri) and the atom

type of the boron atoms of the carborane cages were changed to the C.3 atom type

because Sybyl 7.1 does not provide default parameters for calculations involving

molecules with hexavalent boron atoms of the carborane cluster. This manipulation of

47 boron atom types was described previously by us and was found to be suitable for

docking studies with other carborane-containing molecules.198,199 The three compounds

were then docked into the active site of both available hTK1 crystal structures20,23 using the FlexX module of Sybyl 7.1. However, none of the N5-2OH isomers was found to bind to the dTTP binding site effectively.

Since both hTK1 crystal structures presumably are closed forms of the enzyme, we further analyzed the binding of 3.11.A and the (R)-epimers of 3.11.D and 3.11.E to an

“open” or “semi-open” form of hTK1, as described in the following. The amino acid sequence identity of CaTK, presumably representing an “open” or “semi-open” form, with hTK1 is 39%. Thus, a homology model of hTK1 was generated using CaTK as a template. The initial coordinates of the hTK1 homology model were constructed with

SWISS-MODEL (Version 36.0003).200 The obtained coordinates were then transferred to

Sybyl 7.1 and hydrogen atoms were added. The hydrogen atom positions were minimized

until an RMS of 0.005 kcal·mol-1Å-1 was reached using the Powell method. This

homology model was solvated with water molecules using the Solvent/Solvate option.201

The solvated homology model was minimized until the gradient reached 0.05 kcal·mol-

1Å-1 using the Tripos Force Field. For docking studies, the active site was generated by

selecting the same amino acid residues that are located within a radius of 12.0 Å from

dTTP in the hTK1 crystal structure (PDB ID:1W4R).20

(A) (B)

Figure 3.7. (A) hTK1 crystal structure (PDB ID: 1W4R) with dTTP (B) hTK1 homology model docked with 3.11.A (C) hTK1 homology model docked with (R)-epimer of 3.11.D (D) hTK1 homology model docked with (R)-epimer of 3.11.E

a: lasso domain, b: loop connecting the β2 and β3 strands, c: extended cleft between the lasso domain and the loop connecting the β2 and β3 strands

49 As shown in Figure 3.7.A and 3.7.B, the difference in the distance between the

loop connecting the β2 and β3 strands (b) and the “lasso” domain (a) in the crystal

structure of hTK1 and the homology model of hTK1 is substantial. We acknowledge the

limitations of docking studies involving a homology model.202 Eventually, only an in-

depth X-ray crystallographic analysis can reveal realistic dimensions of open and closed forms of hTK1. Nevertheless, in contrast with the hTK1 crystal structures, docking of compounds 3.11.A and (R)-epimers of 3.11.D and 3.11.E into the homology model of hTK1 revealed an interesting binding pattern. The bulky carboranyl side chain at N-3 position of 3.11.A is oriented towards the extended gap (c) between the loop connecting the β2 and β3 strands (b) and the “lasso” domain (a).

We hypothesize that this extended gap allows effective binding of 3.11.A to the active site of the open form of hTK1 and that partial closure of the lasso due to binding of

3.11.A will eventually result in an orientation of the 2´-deoxyribose portion that allows for an effective transfer of the γ-phosphate from ATP to the 5´- or 3´-hydroxyl group of 3.11.A while the carborane cluster of 3.11.A is relocated to the enzyme surface through unfolding and extension of the pentylene spacer.

This model accounts for the efficient phosphorylation of 3.11.A by hTK1 (Table

3.2). It also provides an explanation for the apparent lack of steric factors on the phosphorylation of various N5-2OH isomers by hTK1 (Table 3.2), since the carborane cage, which is the center of all steric alterations, is located towards the surface of the enzyme (Figure 3.7.A through 3.7.D). The (R)-epimer of m-carboranyl N5-2OH (3.11.D) docked in a similar way to the hTK1 homology model as 3.11.A. However, in the case of the (R)-epimer of 3.11.E, the dihydroxypropyl group attached to p-carborane projects

50 away from the active site of hTK1 (Figure 3.7.D) while the same group attached to o-

carborane in 3.11.A apparently folds back to the enzyme surface. The specific orientation of the dihydroxypropyl group in the (R)-epimer of 3.11.E may cause less steric hindrance

during the closure of the “lasso” portion of hTK1 compared with 3.11.A, which could explain the slightly higher relative hTK1 PR (Table 3.2).

3.5. Biological evaluation

3.5.1 Phosphoryl transfer assays (PTAs)

The substrate specificity of hTK1 for all synthesized compounds was evaluated using recombinant human hTK1 in PTAs.203 PRs of the synthesized compounds ranged

from 33% to 45% relative to that of dThd (Table 3.2). 10B-enriched N5-2OH (3.5.B)

showed a PR very similar to that of 11B-abundant N5-2OH (3.5.A). There was no

significant difference between the PRs of the pure epimers 3.11.A and 3.11.B. Therefore,

the stereochemistry at the C-16 position apparently did not affect the phosphorylation by

hTK1. The PRs of the m- and p-carboranyl isomers of N5-2OH (3.11.D and 3.11.E) were

also similar to that of the o-carboranyl N5-2OH (3.11.C). These results demonstrated that

the dihydroxylpropyl group as well as the carborane cage is most likely located outside of

the active site of hTK1, which is also supported by the modeling of N5-2OH binding to hTK1, as discussed in Chapter 3.4. It appears that the dihydroxylpropyl group attached to the carborane cage of the N5-2OH series contributes only to their hydrophilicity but does not affect their binding to hTK1. It should be warranted that pure epimers of 3.11.D and

3.11.E are synthesized to prove our hypothesis. The hTK1 PRs of the N5-2OH series were generated in collaboration with Dr. Staffan Eriksson and coworkers.

51 3.5.2. In vivo studies

Since no significant difference in hTK1 PRs among the synthesized N5-2OH

series was observed, 11B-abundant N5-2OH (3.5.A) was chosen for in vivo biodistribution studies to validate its activity. For these studies, the F98 glioma cell line was selected to induce brain tumors in rats because the biological behavior of intracerebral tumors produced in this way simulates that of human GBM, as discussed in Chapter 2. Also, substrate specificities of rat TK1 are very similar to those of hTK1.151

F98 glioma cells were stereotactically implanted into the right cerebral

hemisphere of Fisher rats. Fourteen days later, two compounds (3.5.A and BPA) were

administered by convection-enhanced delivery (CED). CED can delivery drugs to large

regions of the brain by applying a pressure gradient to establish bulk flow during

interstitial infusion without significant functional or structural damage to the brain. N5-

2OH (3.5.A) (78 µg of boron) was solubilized in 10 µL of aqueous 50% DMSO solution

and BPA-fructose complex (78 µg of boron) in PBS. The rats were euthanized at 1 and

2.5 h after CED. Boron concentration of the collected tissue and blood was determined by direct current plasma-atomic emission spectroscopy (DCP-AES).183 Table 3.3 summarizes the boron biodistribution of 3.5.A and BPA in F98 glioma-bearing rats. N5-

2OH (3.5.A) exhibited high T/Br ratios (> 8) at 1 h and 2.5 h after CED administration.

In contrast, the retention ratio of BPA in tumor to normal brain or blood was much lower, in particular after 2.5 h (< 2).70

This work was carried out in collaboration with Drs. Rolf F. Barth and Weilian Yang.

Boron concentration (µg/g) T/Br Drug Time (h) Ipsilateral Contralateral a Tumor Blood ratio brain brain 3.5.A 1.0 41 ± 11 5 ± 4 < 0.5 < 0.5 8.2 BPA 1.0 68 ± 18 19 ± 14 4 ± 2 4 ± 4 3.6 3.5.A 2.5 16 ± 2 2 ± 2 < 0.5 < 0.5 8.0 BPA 2.5 24 ± 8 15 ± 8 8 ± 4 4 ± 2 1.6

Table 3.3. Boron biodistribution in F98-glioma-bearing rats at 1 and 2.5 h after administration of BPA and N5-2OH by CED.70

a The tumor to brain ratio was calculated from boron concentration of tumor and ipsilateral brain.

3.5.3. Neutron irradiation studies

Since N5-2OH (3.5.A) showed promising biodistribution in rats bearing

intracerebral F98 glioma, 10B-enriched N5-2OH (3.5.B) was used for neutron irradiation

studies in the same rodent/tumor model at the Massachusetts Institute of Technology

(MIT) Nuclear Research Laboratory (Cambridge, MA). F98 glioma-bearing rats were

divided into three groups of 7-9 animals each, as follows: Group I, 500 µg of 3.5.B,

administered intracerebrally (i.c.) by CED, followed by neutron irradiation; Group II

received 50% DMSO alone by CED, followed by neutron irradiation; and Group III

received 500 µg of 3.5.B by CED without neutron irradiation. Biodistribution of 3.5.B

was determined in a group of 4 rats that received 3.5.B, as per Groups I and III, but these

animals were euthanized 24 hours later and samples of tumor, blood and brain were analyzed for boron content by DCP-AES and the corresponding boron concentrations

were 17.3 ± 4.3 µg/g for tumor and < 0.5 µg/g for both normal brain and blood. 53

Group I (3.5.B & Group II (DMSO & Group III (3.5.B & no Time neutron irradiation) neutron irradiation) irradiation) 3 weeks 9a/9b 8/8 6/7 4 weeks 9/9 5/8 0/7 5 weeks 5/9 1/8 0/7 6 weeks 2/9 0/8 0/7 Mean ± SD 37 ± 7 days 31 ± 3 days 25 ± 2 days

Table 3.4. Survival of F98 glioma-bearing rats in preclinical neutron irradiation studies

a designates the number of surviving animals per group b designates the total number of animals per group

Survival data are summarized in Table 3.4. Animals that received 3.5.B, followed

by BNCT, had a mean survival time of 37 ± 7 days compared to 31 ± 3 days for

irradiated controls and 25 ± 2 days for untreated controls, and the statistical difference between these groups was significant (p < 0.02). These preliminary survival data are encouraging since they were carried out without optimizing formulation, dosing and delivery of 3.5.B. Furthermore, it should be noted that the plasma concentrations of dThd, which competes with 3.5.B at the active site of hTK1, are 10 times lower in humans than in rodents.204 therefore, BNCT with 3.5.B could be significantly more effective in

humans than in rodents.

Neutron irradiation experiments were carried out in collaboration with Drs. Rolf F.

Barth and Weilian Yang (OSU) and Drs. Peter J. Binns, Kent J. Riley and Jeffrey A.

Coderre (MIT).

54 3.6. Summary and conclusions

Two feasible synthetic routes for the preparation of 10B-enriched N5-2OH were

developed and 10B-enriched N5-2OH was evaluated as a boron-delivery agent for BNCT

in pilot neutron irradiation experiments. Treatment of rats, bearing intracerebral F98

glioma, with 10B-enriched N5-2OH (3.5.B) and neutron irradiation resulted in prolonged

survival compared to control groups of animals, indicating that 10B-enriched N5-2OH has

a significant potential as a boron-delivery agent for BNCT of GBM. Stereochemical- and

geometrical N5-2OH isomers were synthesized and their specificities as substrate for

hTK1 were evaluated to complete the SAR studies of the N5-2OH series. A

computational model for the binding of N5-2OH to the active site of hTK1 was developed. hTK1 PRs and docking studies indicated that the dihydroxypropyl group

attached to the carborane cluster of the N5-2OH isomers may only function as a

hydrophilicity-enhancing structural element, but does not increase the binding affinity to

the active site of hTK1. Overall, the studies described in Chapter 3 identified 10B- enriched N5-2OH as one of the most promising novel BNCT agents.

The work described in Chapter 3 was partially published in Cancer Research

(Barth et al., 2004, 64, 6287-6295).

3.7. Outlook: Challenges ahead

Although the pilot neutron irradiation studies with 10B-enriched N5-2OH (3.5.B) produced very promising results, further structural improvement of 3CTAs is warranted.

A major problem of N5-2OH is its low solubility in water necessitating the use 50% aquesous DMSO for in vivo studies (see Chapters 3.4 and 3.5). Approaches to overcome

55 the low water-solubility of N5-2OH will be addressed in Chapter 4 in more detail. The

second concern is the real-time pharmacokinetics of 10B-enriched N5-2OH in clinical

studies since the measurement of tissue boron concentration via DCP-AES is only suitable to a limited extent for this purpose. One solution would be the introduction of a radioactive isotope such as 123I into 3CTAs, which would allow real-time imaging via

Single Photon Emission Computed Tomography (SPECT). Attachment of other iodine

isotopes, such as 125I or 131I, could even lead to the use of 3CTAs in the conventional

radiation therapy.205

O O N N OH OH a HO OH HO N O OH N O O O I OH OH 3.12.A

O O N OH N OH HO N O OH HO N O OH O O I I I I I OH OH C B-H 3.12.B 3.12.C B

Scheme 3.6. Iodination of N5-2OH

Reagents and conditions: (a) I2, AlCl3, CH2Cl2, reflux, 48 h.

A major problem associated with the biomedical applications of drugs labeled with radioactive iodines is susceptibility to dehalogenation. Release of radioiodine from drugs leads to the accumulation of significant quantities of radioactive iodine in the 56 thyroid and stomach.205,206 Iodine linked to boron atoms of the carborane cage in 3CTAs

may overcome this problem of cleavage of carbon-iodine bonds because the B-I bond in

the carborane cage in 3CTAs is expected to be less susceptible enzymatic and hydrolytic

cleavage than the C-I bond.207,208

Therefore, we have conducted the pilot experiement shown in Scheme 3.6. Direct iodination of N5-2OH was carried out with I2 in dichloromethane in the presence of

165,166 AlCl3 at 40 °C for 48 h (Scheme 3.6). Products (3.12.A, 3.12.B, and 3.12.C) were isolated by analytical HPLC. The structures of 3.12.A, 3.12.B, and 3.12.C were confirmed by 1H-NMR, HRMS, and 11B-NMR. Interestingly, another set of three iodinated compounds separated by silica gel chromatography were identified as the thymine derivatives of 3.12.A, 3.12.B, and 3.12.C. This demonstrated that the glycosidic bond is to some extent susceptible to cleavage under the reaction conditions. To our knowledge, this is the first report on the direct iodination of the closo-carborane cage in a bioactive molecule, although the exact positions of the iodine substituents at the carborane cages in 3.12.A, 3.12.B, and 3.12.C have not yet been assigned.

Eventually, the success of N5-2OH iodination may facilitate pharmacokinetic studies of 10B-enriched N5-2OH. By applying the route described in Scheme 3.6,

radioactive iodine can be attached directly to the carborane cage by using radioactive I2.

Alternative radioiodination of 3.12.A, 3.12.B, and 3.12.C may be achieved by palladium- catalyzed nucleophilic substitution using radioactive NaI (e.g Na123I) and Herrmann’s

catalyst.209,210

57 CHAPTER 4

INVESTIGATION OF BIOCHEMICAL, BIOLOGICAL, AND CHEMICAL

PROPERTIES OF ZWITTERIONIC NIDO CARBORANE

4.1. Background and rationale

Research activities on discovering novel BNCT agents in our laboratories have

focused on the synthesis and biological evaluation of N5-2OH, as discussed in Chapter 3.

Although the evaluation of N5-2OH in preclinical BNCT studies produced very

promising results,70,181 a major drawback of this 3CTA is the lack of water-solubility,

precipitated by the high hydrophobicity of its closo o-carborane cluster.164 Several

strategies have been explored to improve the water-solubility of 3CTAs including the

introduction of multiple hydroxyl groups,211 PEGylation,61 and formation of anionic or

zwitterionic nido-carboranyl 3CTAs, the latter being the topic of this Chapter.

Biomolecules substituted with negatively charged nido-carborane are usually

water-soluble because of the anionic character of the nido-carborane cage.207 In addition, the transformation of closo carboranyl biomolecules into their nido counterparts has only a minimal effect on their overall structure, and thus, provides a means of manipulating physicochemical properties while maintaining biological activities. However, it seems

58 unlikely that such anionic structures can cross cell membranes effectively via passive

diffusion due to their highly hydrophilic character. Therefore, several 3CTAs containing a

+ zwitterionic NH3 -nido m-carborane were synthesized and evaluated biologically

(Chapter 4.2).

+ The basis for the unique physicochemical properties of zwitterionic NH3 -nido m- carboranyl 3CTAs is the intramolecular charge compensation between the positively charged ammonium group and the negatively charged nido m-carborane cage, presumably leading to structures with balanced hydrophilicity/hydrophobicity properties. A few nido o- carboranes that were substituted with charge-compensating sulfonium, pyridinium, or phosphonium group at a boron atom of the cluster have been reported.212-214 Therefore,

+ we investigated an alternative zwitterionic NH3 -nido m-carborane system both experimentally and theoretically (Chapter 4.3).

4.2. Synthesis and biological evaluation of zwitterionic nido 3CTAs for BNCT

4.2.1. Synthesis

+ Zwitterionic NH3 -nido m-carborane containing 3CTAs were synthesized as

shown in Scheme 4.1. Starting material 4.1 was prepared on a gram scale applying a

procedure previously reported by Kahl and coworkers.121 The tosylates used for the

alkylation of 4.1 were either purchased [ethyleneglycol di-p-tosylate, di(ethyleneglycol)

di-p-tosylate] or prepared from 1,3-propanediol, 1,4-butanediol, 1,5-pentanediol, and 1,6-

hexanediol as described previously.215,216 Alkylation of 4.1 with the alkanediol di-p-

tosylate and di(ethyleneglycol) di-p-tosylate using 2.2 equivalents of n-BuLi in benzene

afforded the m-carboranyl derivatives 4.2-4.7 in 14-45% yield. The relatively low yield

59 of 4.7 (14%) is probably due to both the electron-withdrawing effect of the oxygen atom

in the diethylene ether spacer and the low solubility of di(ethyleneglycol) di-p-tosylate in

benzene.

O X NHBoc N H NHBoc TsO X NHBoc a b HO N O O

4.1 4.2: X=(CH2)2 OH 4.8: X=(CH2)2 4.3: X=(CH2)3 4.9: X=(CH2)3 4.4: X=(CH2)4 4.10: X=(CH2)4 4.5: X=(CH2)5 4.11: X=(CH2)5 4.6: X=(CH2)6 4.12: X=(CH2)6 4.7: X=(CH2)2O(CH2)2 4.13: X=(CH2)2O(CH2)2

O O H X NH2 X NH N N 3 c d HO N O HO N O O O

OH OH 4.14.A: X=(CH2)2 4.14.B: X=(CH2)2 4.15.A: X=(CH2)3 4.15.B: X=(CH2)3 4.16.A: X=(CH2)4 4.16.B: X=(CH2)4 4.17.A: X=(CH2)5 4.17.B: X=(CH2)5 4.18.A: X=(CH2)6 4.18.B: X=(CH2)6 4.19.A: X=(CH2)2O(CH2)2 4.19.B: X=(CH2)2O(CH2)2

Scheme 4.1. Synthesis of zwitterionic nido 3CTAs and the neutral closo counterparts a o Reagents and conditions: (a) Alkanediol di-p-tosylates, n-BuLi, benzene, 10 C, 0.5-1 h; (b) dThd, K2CO3, o o DMF/acetone (1:1), 50 C, 48 h ; (c) CF3COOH, CH2Cl2, rt, 24 h; (d) TBAF, THF, 70 C, 1-2 h.

Condensation of compounds 4.2-4.7 with dThd was carried out in the presence of

K2CO3 in DMF/acetone (1:1) at 50 °C for 48 h to provide compounds 4.8-4.13 in 50-85% yield. The tert-butoxycarbonyl (Boc) group was subsequently removed with 60 trifluoroacetic acid (TFA) in dichloromethane at room temperature for 24 h to give the

neutral closo m-carboranyl 3CTAs (4.14.A-4.19.A) in 69-87% yield. Treatment of

160 4.14.A-4.19.A with tetrabutylammoniumfluoride hydrate (TBAF•H2O) in THF at 70

°C for 1-2 h and subsequent acidic workup provided the corresponding zwitterionic nido

3CTAs (4.14.B-4.19.B) in 50-70% yield. Formation of the nido carboranes was monitored by IR, which showed the disappearance of the typical B-H band of closo carboranes around 2590 cm-1 and emergence of the B-H band of nido-carboranes around

2530 cm-1.

4.2.2. Analysis

Analytical C18- and C8 reversed phase HPLC (RP-18 and RP-8) was carried out

with compounds 4.14.A/B-4.19.A/B for the following reasons: (i) to verify their purity,

(ii) to obtain amounts of 1-3 mg in highly purified form for PTAs (Figure 4.2 and Table

4.1), and (iii) to attain retention times as measures of their lipophilicities (Table 4.1).

Water/methanol gradient (100:0 to 70:30 over 10 min, from 70:30 to 90:10 over 20 min, and from 90:10 to 0:100 over 10 min) with a flow rate of 1 mL/min was applied for all target compounds using RP-8 and RP-18. The obtained retention times of 4.14.A/B-

4.19.A/B ranged from 14.3 to 24.8 min while those of AZT and nido o-carboranyl 4.2062

(see Figure 4.2 for the structure) were 12.2 and 11.8 min, respectively. Compound 4.20 and AZT were selected as control compounds as representatives for pure anionic nido

3CTAs and established therapeutic nucleoside prodrugs, respectively. It should be noted that all zwitterionic nido 3CTAs had longer retention times than AZT, which is known to penetrate cell membranes mainly via passive diffusion.217

61 TK1 TK2 RP-18 retention Compound (10 µM) (100 µM) times 4.14.A 71.9 ± 5.1 < 0.1 19.01

4.14.B 89.4 ± 1.2 < 0.1 14.32a

4.15.A 58.2 ± 2.5 < 0.1 19.08

4.15.B 64.6 ± 4.0 < 0.1 14.96

4.16.A 58.0 ± 5.3 0.4 20.81

4.16.B 63.8 ± 3.1 0.1 15.38

4.17.A 45.1 ± 2.6 0.4 22.65

4.17.B 50.7 ± 1.6 1.2 16.36

4.18.A 14.2 ± 2.1 0.7 24.76

4.18.B 45.4 ± 2.0 1.3 17.77

4.19.A 48.4 ± 1.6 0.9 18.08

4.19.B 75.1 ± 0.6 0.5 14.48

4.20 44.8 ± 1.7 N/A 11.78

N5-2OHb 41.0 ± 5.0 < 0.1 22.54

AZTc 52.0 4.0 12.22

dThd d 100.0 100.0 8.72

Table 4.1. hTK1/TK2 PRs and RP-18 retention times of 3CTAs, AZT, and dThd

a Two peaks were observed. The average value of both peaks (14.21 min and 14.43 min) was inserted. b,c hTK1 and TK2 PRs were reported previously.43,59 d PR for dThd were set to 100. Mean ± SD values are based on three experiments for recombinant hTK1 and one experiment for TK2. N/A: Not determined

62 Interestingly, during RP-18 HPLC analysis of compound 4.14.B, two close peaks

were observed at 14.21 and 14.43 min. RP-8 HPLC analysis produced a similar pattern

with peaks at 14.49 and 14.65 min. This was not the case for 4.14.A-4.19.A and 4.15.B-

4.19.B. The compounds corresponding to these peaks were isolated and analyzed by

NMR and HRMS. While HRMS data for both compounds were indicative for the

structure of 4.14.B, several differences were observed in the 1H-NMR and 13C-NMR spectra, in particular for the signals of the C-1´ proton (6.27 ppm and 6.36 ppm) and the

N-CH2 protons, as shown in Figure 4.1. Similar chemical shifts patterns were not

observed for 4.14.A-4.19.A and 4.15.B-4.19.B.

Figure 4.1. Partial 1H-NMR spectra of two pure epimers of 4.14.B 63 Degradation of closo m-carborane into its nido m-counterpart introduces a chiral

element,158-160,174 which in the case of the zwitterionic 3CTAs 4.14.B-4.19.B results in a

mixture of two epimers. The close proximity of the bulky nido carborane cluster to the dThd scaffold in 4.14.B could cause additional steric constraint emphasizing structural differences between both epimeric structures (Figure 4.1). However, this splitting pattern might also derive from an atropisomeric effect due to hindered rotation of the single bonds between nido carborane cluster and dThd scaffold. If the obtained fractions from

HPLC were two pure atropisomers, increasing the temperatures should promote their interconversion. However, we did not observe any changes in 1H-NMR spectra of both

forms that were obtained before and after 1 h-reflux in methanol, indicating the formation

of pure epimers (Figure 4.1). Further analysis, ideally through X-ray crystallography,

would be required to substantiate this hypothesis.

4.2.3 Phosphoryl transfer assays (PTAs)

The β-autoradiogram depicted in Figure 4.2 shows the monophosphate (MP)

products of the neutral closo carboranyl 4.15.A, 4.17.A, and N5-2OH, anionic nido

carboranyl 4.20, zwitterionic nido carboranyl (4.15.B and 4.17.B), and dThd (‘b’). In

previous studies with similar 3CTAs, combined α/β-autoradiography provided evidence

that the observed intense spots with high Rf values (‘a’) are typical for MPs of

3CTAs.59,61,62 The migration of the MPs also provides information on hydrophilicity

differences between the anionic nido-, zwitterionic nido-, and neutral closo 3CTAs. It is

obvious that the Rf values of the MPs of the zwitterionic nido 3CTAs (4.15.B and 4.17.B) are between the MPs of neutral closo- and anionic nido 3CTAs. 64

Figure 4.2. Phosphorylation of neutral closo carboranyl 4.15.A, 4.17.A, and N5-2OH, anionic nido carboranyl 4.20, zwitterionic nido carboranyl 4.15.B and 4.17.B, and dThd by recombinant hTK1.

The reaction products were separated by PEI-cellulose TLC and visualized by 32P-autoradiograhy. (a) MP products of the compounds 4.20, 4.15.B, 4.15.A, 4.17.B, 4.17.A, and N5-2OH. (b) dTMP.

The exact PRs of compounds 4.14.A/B-4.19.A/B relative to that of dThd with hTK1 and TK2 are summarized in Table 4.1. The hTK1 PRs of compounds 4.14.A-

4.19.A (neutral closo 3CTAs) ranged from 71.9 ± 5.1% to 14.2 ± 2.1% and those of compounds 4.14.B-4.19.B (zwitterionic nido 3CTAs) ranged from 89.4 ± 1.2% to 45.4 ±

2.0% (Table 4.1). The hTK1 PR of 4.14.B (89.4 ± 1.2%) is the highest among the

reported 3CTAs. Preliminary enzyme kinetic analysis of 4.14.B resulted in a kcat/KM value

65 of ~ 49% relative to that of dThd. The hTK1 PRs of the zwitterionic nido m-carboranyl series 4.14.B-4.19.B were 5-30% higher than those of the corresponding neutral closo m- carboranyl series 4.14.A-4.19.A. The hTK1 PRs decreased in both series with increasing spacer length almost in a linear fashion except compounds 4.19.A/B, which have diethylene ether spacers.

As shown in Table 4.1, the hTK1 PRs of 4.14.A/B-4.19.A/B are inversely proportional to their RP-18 retention times, and thus, to their lipophilicity. In the cases of zwitterionic compound 4.19.B, having a diethylene ether spacer, the RP-18 retention time

(14.48 min) and the hTK1 PR (75%) are more comparable with those of compounds

4.14.B (14.31 min and 89 %) and 4.15.B (14.96 min and 65%), having ethylene- and propylene spacers, rather than those of 4.17.B (16.36 min and 51%) and 4.18.B (17.77 min and 45%) with pentylene- and hexylene spacers comparable in lengths to the diethylene ether spacer in 4.19.B. Therefore, it appears that the determining factor for their hTK1 PRs of zwitterionic nido 3CTAs is their lipophilicity rather than their spacer lengths. However, we cannot exclude the possibility that the oxygen atom in the spacer of

4.19.B affects the hTK1 PR through specific hydrogen-bonding interactions in the substrate binding site of hTK1.

Only at high substrate concentrations of 100 µM, compounds 4.16.A/B-4.19.A/B, having butylene-, pentylene-, hexylene-, and diethylene ether spacers, appeared to be phosphorylated to a limited extent by TK2 with PRs ranging from 0.1-1.3%, while

4.14.A/B-4.15.A/B with shorter ethylene- and propylene spacers apparently were not substrates of TK2.

66 Overall, zwitterionic nido 3CTAs showed a unique hydrophilcity/hydrophobicity balance between neutral closo 3CTAs, which are not sufficiently soluble in water for preclinical studies, and anionic nido 3CTAs, which are presumably too hydrophilic to

cross cell membranes via passive diffusion. Therefore, zwitterionic nido 3CTAs may be

able to penetrate cell membranes by passive diffusion while having increased water

solubility compared with N5-2OH.

All PRs were obained in collaboration with Dr. Staffan Eriksson and coworkers.

4.2.4. In vivo studies

Although compound 4.14.B has better enzymatic properties than compound

4.15.B, the latter was selected for preliminary in vivo biodistribution studies in mice

bearing subcutaneous (s.c.) L929 (wt) or L929 TK1 (-) tumors because it was available in

sufficient quantities. BPA and N5-2OH were used as reference compounds. All

compounds were injected intratumorally (i.t.) twice with a 2-h interval. Each injection

included compound quantities equivalent to 50 µg boron. This procedure mimics to some

degree the condition of CED to brain tumors, which is the proposed route of

administration for 3CTAs (Chapter 3.5.2). Boron concentrations in L929 (wt) tumors

were 21.7 ± 9.1, 29.8 ± 7.4, and 4.0 ± 2.1 µg/g tumor for 4.15.B, N5-2OH, and BPA,

respectively, at 2 h following the 2nd injection (Figure 4.4). The corresponding values for

the L929 TK1 (-) tumors, were 7.0 ± 5.9, 12.1 ± 7.7, and 5.8 ± 3.1 µg/g tumor. Boron

concentrations in blood, skin, and liver were < 0.5 µg/g tissue in both tumor/rodent

models. The values for 4.15.B and N5-2OH in L929 (wt) tumors were comparable and in the concentration range necessary for BNCT, while those for BPA were significantly 67 lower. The values for nucleoside analogues 4.15.B and N5-2OH in L929 TK1 (-) tumors were 3.1 and 2.5 times lower than those in L929 (wt) tumors. However, the amino acid

BPA was 1.5 times higher in L929 TK1 (-) tumors than in L929 (wt) tumors.

Boron concentration (µg/g) Compound L929 (wt) L929 TK1 (-)

4.15.B 21.7 ± 9.1 7.0 ± 5.9

N5-2OH 29.8 ± 7.4 12.1 ± 7.7

BPA 4.0 ± 2.1 5.8 ± 3.1

Table 4.2. Tumor uptake of 4.15.B, N5-2OH, and BPA in mice bearing s.c. implants of L929 (wt) and L929 TK1 (-) tumor.

Compound quantities equivalent to a total of 100 µg boron were given in two intratumoral injections in a 2- h interval. Boron concentrations in tissues were determined at 2 h following the second injection by DCP- AES.183 Each data point represents the arithmetic mean ± SD of 4 mice.

These results indicated that the uptake of 4.15.B and N5-2OH is related to some form of nucleoside metabolism, presumably intracellular entrapment through 5´- monophosphorylation by hTK1. However, we cannot exclude the possibility that cellular efflux mechanisms specific to L929 TK1 (wt) and L929 TK1 (-) may contribute to the observed biodistribution patterns.

As expected, the zwitterionic compound 4.15.B showed improved water solubility compared to N5-2OH. In order to dissolve compound quantities equivalent to 50 µg

boron, 15 µL of DMSO/H2O (70:30) were required for N5-2OH while 4.15.B was

68 dissolved completely in 10 µL of DMSO/H2O (24:76), indicating significantly increased

water solubility properties of 4.15.B.

The in vivo studies described in Chapter 4.2.4 were carried out in collaboration

with Dr. Rolf F. Barth and coworkers.

4.2.5. Summary and Conclusions

+ Six zwitterionic NH3 -nido m-carborane containing 3CTAs and their corresponding neutral NH2-closo m-carborane substituted counterparts were synthesized.

Their PRs were evaluated with recombinant hTK1 and TK2. The obtained hTK1 PRs

ranged from 89% to 14% relative to dThd and appeared to be dependent mainly on the

lipophilicity of the compounds although structural contributions, such as the spacer

length between carborane cluster and dThd scaffold, may also be of importance. The

hTK1 PR of zwitterionic compound 4.14.B (89%) was the highest determined for any

3CTA. HPLC RP-18 retention times indicated that the lipophilicity of zwitterionic

compounds such as 4.14.B and 4.15.B may be sufficient for passive diffusion through

cell membranes. One zwitterionic compound 4.15.B was preferentially taken up in L929

(wt) tumors implanted in nude mice and showed significantly improved water solubility

compared with N5-2OH. Overall, zwitterionic nido 3CTAs appears to have similar or

even improved biological properties than N5-2OH in combination with improved

physicochemical properties (e.g. water solubility). It should be noted that 4.14.B

presumably has biodistribution and water-solubility properties that are superior to those

of 4.15.B.

69 The work described in Chapter 4.2 was published in the Journal of Medicinal

Chemistry (Byun et al. 2005, 48, 1188-1198) and in Applied Radiation and Isotopes

(Byun et al. 2004, 61, 1125-1130).

4.3. Experimental and theoretical studies of zwitterionic nido m-carborane system

The zwitterionic nido m-carboranyl 3CTAs described in Chapter 4.2 showed balanced hydrophilicity/hydrophobicity properties unlike those of lipophilic closo and

+ hydrophilic nido 3CTAs, demonstrating the usefulness of NH3 -nido m-carborane as a

novel boron moiety in BNCT drug design. Since there has been no previous report on this

specific carborane cage structure, we further investigated some of its synthetic,

spectroscopic, and theoretical features.

4.3.1. Synthesis

H H H H H NHBoc H H H N H N a b H c H

m-carborane 4.1 4.21 4.22

Scheme 4.2. Synthesis of zwitterionic nido m-carborane (4.22)

Reagents and conditions: (a) 1) n-BuLi/THF/CO2; 2) PCl5/toluene; 3) (CH3)3SiN3/toluene, t-BuOH, reflux; (b) CF3COOH, dichloromethane, rt, 12 h; (c) TBAF, THF, 70 °C, 1.5 h.

Compound 4.1 was prepared from m-carborane (1,7-C2B10H12) in 3 steps as

described previously.121 Deprotection of the Boc group was achieved by treatment with

70 TFA at room temperature for 12 h to afford 1-amino-1,7-carborane (4.21) in 85% yield.

Reaction of compound 4.21 with TBAF in THF for 1.5 h at 70 °C afforded zwitterionic nido m-carborane (4.22) in 61% yield.

4.3.2. Structural analysis

Figure 4.3. Structures of 4.21 and 4.22 optimized at the B3LYP/6-31G* level

The bridging hydrogen of compound 4.22, designated as 25H in Figure 4.3, was

observed at -1.99 ppm in 1H-NMR due to nuclear shielding by the electron-rich nido m-

carborane cluster. This phenomenon has been discussed previously by Onak et al.218 The reduced shielding in the electron deficient closo-carborane cluster and increased shielding in the electron-rich zwitterionic nido-carborane affect the chemical shifts of the hydrogen atoms (17H) attached to the carbon at the 9-position of both compounds. Hydrogen peak of 17H in 4.22 appeared at 1.43 ppm while that of compound 4.21 appeared at 3.23 ppm.

71 13C-NMR spectra showed similar patterns. Chemical shifts for the two carbons (9C and

21C) of 4.22 were 32.83 ppm and 53.77 ppm, respectively. In contrast, two peaks of 4.21

were observed in a low-field region (9C: 54.65 ppm, 21C: 90.45 ppm).

Figure 4.4. 11B (proton-decoupled)-11B (proton-decoupled) COSY spectrum of 4.22

72 11B-11B correlation spectroscopy (11B-11B COSY) shown in Figure 4.4, in

combination with common prediction rules for borane or heteroborane clusters,219 provided the complete assignment for the nine boron atoms of compound 4.22.

According to these prediction rules, signals for boron atoms with higher coordination number appear in a high-field region and substitution of boron with carbon in the cage scaffold causes a low-field shift of the boron atom opposite to carbon.219 Based on these

rules, 2B and 3B (Figure 4.3) were expected to appear in a high-field region because both of them coordinate five boron atoms (2B: 1B, 3B, 4B, 6B and 7B, 3B: 2B, 4B, 7B, 8B and 10B). Three boron atoms (5B, 4B and 7B) were expected in a low-field region because 5B coordinates only two boron atoms (8B and 10B), and 4B and 7B are at the position antipodal to 9C and 21C, respectively. Nine boron atoms of compound 4.22 were

assigned via the 11B-11B COSY spectrum shown in Figure 4.4.

IR spectra of compounds 4.21 and 4.22 showed two distinctive stretching bands

for B-H and C-N. B-H stretching bands for 4.21 and 4.22 were at 2595 cm-1 and 2535 cm-

1 respectively. This frequency change is a major spectroscopic feature to distinguish closo

carboranes from their nido counterparts. The C-N stretching band of compound 4.21 was

1611 cm-1, which is very close to the typical C=N stretching band while that of compound 4.22 was 1467 cm-1. Recently, Boyd and coworkers reported exo π-bond

formation between a carbon atom of o-carborane and several π-donor substituents (OH,

212 NH2, NH, and CH2) attached to it. The observed exo C-N distance in 1-amino-2-

phenyl-ortho-carborane was 1.392 Å, which is shorter than that of a typical C-N single

bond (~ 1.47 Å). Therefore, we hypothesized that the C-N bond of compound 4.21 also

has exo C-N π-bonding character.

73 4.3.3. Theoretical calculations

Methods: Theoretical ab initio HF and DFT calculations for compounds 4.21 and

4.22 were performed using the Gaussian 03 program running on an Itanium 2 Cluster at

Ohio Super Computer Center. The structures were optimized at the HF/6-31G*, HF/ 6-

31G**, B3LYP/6-31G*, and B3LYP/6-31G** levels. NMR chemical shift calculations

were performed with the gauge-including atomic orbitals (GIAO) method using the

11 optimized geometries in acetonitrile (CH3CN) solution. Theoretical B-NMR chemical

shifts were computed versus the chemical shift of diborane (16.6 ppm) and then

1 13 converted to the BF3•Et2O scale. H-NMR and C-NMR chemical shifts were referenced to tetramethylsilane (TMS) standard.

Table 4.3 lists the total energies of the optimized structures of 4.21 and 4.22 at the

HF and DFT levels and Table 4.4 summarizes the charge distributions of the heavy atoms and the bridging hydrogen of 4.21 and 4.22.

Compound HF/6-31G* HF/6-31G** B3LYP/6-31G* B3LYP/6-31G**

4.21 -384.6639 a.u. -384.6836 a.u. -387.4690 a.u. -387.4869 a.u.

4.22 -360.4853 a.u. -360.5080 a.u. -363.1490 a.u. -363.1695 a.u.

Table 4.3. Total energies of optimized structures 4.21 and 4.22

74 4.21 4.22 Atom Mulliken charge APT charge Mulliken charge APT charge 1B 0.098 0.117 -0.015 0.185 2B 0.032 0.061 -0.001 -0.035 3B 0.035 0.062 0.122 -0.001 4B 0.023 0.162 0.023 0.248 5B 0.081 0.171 0.088 0.260 6B 0.044 0.150 0.012 0.225 7B 0.005 0.169 0.031 0.272 8B 0.042 0.140 0.019 0.142 9C -0.331 -0.199 -0.311 -0.319 10B 0.050 0.114 0.002 0.122 21C -0.038 0.167 -0.097 -0.125 22B 0.066 0.193 - - 24N -0.605 -0.487 -0.560 -0.198 26H -0.002 -0.120 0.046 -0.047

Table 4.4. Mulliken- and APT charges of heavy atoms and the bridging hydrogen atom for 4.21 and 4.22 at the B3LYP/6-31G** level.

Gaussian 03 program provides two types of atomic charge populations as default settings: Mulliken and atomic polar tensor (APT). As shown in Table 4.4, there is a significant difference of atomic charge distributions between Mulliken charge and APT charge for compounds 4.21 and 4.22. The boron atoms 5B and 22B, which are attached to the carbon atoms of 4.21, are expected to have the highest positive charge because they are susceptible to removal by fluoride treatment. APT charge calculation showed that these two boron atoms (5B and 22B) of 4.21 were indeed those with the highest charges

75 (0.171 and 0.193) while Mullikan charge calculations identified 1B (0.099) as the most

positively charged boron atom. Distances between 21C and 24N of optimized structures

4.21 and 4.22 at the B3LYP/6-31G** level were 1.4309 Å and 1.5078 Å, respectively,

indicating exo C-N π-bond character of compound 4.21.

Theoretical chemical shifts of nine boron atoms for 4.22 were obtained using

GIAO/11B-NMR calculations at the HF/6-31G*, HF/6-31G**, B3LYP/6-31G*, and

B3LYP/6-31G** levels. Table 4.5 summarizes the experimental and theoretical 11B-

NMR chemical shifts of compound 4.22. NMR chemical shifts obtained experimentally showed strong correlations (r2: 0.9957) with those calculated using DFT (B3LYP/6-31G*

and B3LYP/6-31G**) methods.

Atom Experimentala HF/6-31G* HF/6-31G** B3LYP/6-31G* B3LYP/6-31G** 4B 0.82 2.58 3.45 -4.08 -2.81 5B -2.82 0.20 1.02 -5.82 -4.55 7B -3.6 -7.21 -6.29 -6.30 -5.01 10B -15.75 -16.23 -15.12 -17.44 -16.14 8B -19.53 -17.71 -17.11 -21.94 -20.66 6B -20.07 -18.69 -17.77 -22.55 -21.30 1B -20.07 -22.50 -21.65 -23.26 -21.97 3B -31.95 -32.50 -31.55 -33.34 -32.07 2B -33.59 -35.08 -33.95 -35.39 -34.10 r2 0.9646 0.9659 0.9957 0.9957 Standard deviation 2.4824 2.4382 0.8663 0.8659

Table 4.5. Experimental and theoretical (GIAO) 11B-NMR chemical shifts of 4.22

a Experimental NMR chemical shifts were recorded in deuteriated CH3CN solution at 25 °C.

76 4.3.4. Observations

Figure 4.5. Proton-decoupled 11B-NMR spectra of compound 4.22 in deuteriated acetone solution recorded as a function of time a The 0 min spectrum was recorded right after dissolving 4.22 in deuteriated acetone.

During 11B-NMR studies with compound 4.22 in deuteriated acetone

(CD3COCD3), we observed time-dependant disappearance and emergence of signals, as shown in Figure 4.5. Interestingly, we noticed that the solution of 4.22 in CD3COCD3 at

180 min contained a UV active compound that could be detected at 254 nm via TLC analysis. To explore potential solvent effects on NMR chemical shifts and the formation of an UV active component, the capacity of various solvents (acetone, methylethylketone, 77 acetonitrile, benzene, chloroform, diethylether, dimethylsulfoxide, ethylacetate, hexane, methanol, tetrahydrofuran, and water) to form an UV-active complex with 4.22 was

studied by UV/TLC analysis. Only acetone and methylethylketone (CH3CH2COCH3)

appeared to interact 4.22 to form UV-active species. Indeed, reaction of 4.22 with

acetone at room temperature for 6 h, and subsequent removal of acetone under reduced

pressure yielded the iminium-containing compound 4.23 in a quantitative yield (Scheme

4.3). The chemical structure of 4.23 was confirmed by 1H-, 13C-, 11B-NMR and HRMS.

Compound 4.23 was fairly stable in solvents such as methanol, benzene, and

dichloromethane although a small amount (~ 10%) of 4.22 was detected after 24 h at

room temperature.

H H H H H N O H N H H

4.22 4.23

O H H N 4.22 H benzene

4.24 OCH3

OCH3 H H H H 4.22 N OCH3 benzene H O OCH3

4.25

Scheme 4.3. Preparation of iminium-containing compounds (4.23-4.25) from 4.22 78 The UV absorption spectra (Figure 4.6) of 10-5 M solutions of compounds 4.22

and 4.23 in methanol showed bands at the 220 nm and 253 nm regions. Only the 220 nm

band is a typical characteristic for the 3D σ-aromatic system of carboranes.154 The band at

253 nm had significant intensity only in the case of 4.23 whereas it was reduced to merely a shoulder in 4.22. A similar UV absorption shift was reported previously for the

σ-aromatic closo-p-carborane cluster when conjugated with π-aromatic benzene.220 To

the best of our knowledge, however, compound 4.23 is the first example of stable

conjugation of a 3D σ-aromatic system of a nido carborane cluster with a non-aromatic iminium group, resulting in a significant alteration in the UV absorption. We hypothesize that an electron delocalization from the negatively charged nido m-carborane cluster to the iminium double bond is responsible for this bathochromic shift.

Figure 4.6. UV absorption spectra of compound 4.22 and 4.23 (10-5 M solutions in MeOH)

79 Molecular Orbital (HOMO/LUMO) calculations (Figure 4.7) for 4.22 and 4.23 at

the B3LYP/6-31G** level supported the bathochromic shift of 4.22 and 4.23 observed in

UV experiment. As shown in Figure 4.7, in the case of the conjugated molecule 4.23,

alternating-phase orbitals with similar sizes stemming from iminium nitrogen group and

the nido carborane cluster overlapped completely. In particular, the HOMO of 4.23

showed a pattern of overlapping orbitals similar to that of benzene. In contrast, there is no

significant overlap of alternating-phase orbitals between carborane cluster and nitrogen in

4.22. The energy gap between HOMO and LUMO of compound 4.23 was 4.98 eV, which

is notably smaller than that of compound 4.22 (5.52 eV). It is well known that

conjugation increases with a decreasing the energy gap between HOMO and LUMO decreases.221

Figure 4.7. Diagrams of HOMO and LUMO for 4.22, 4.23, and benzene

80 4.3.5. Applications of novel zwitterionic nido m-carborane cluster

As mentioned in Chapter 4.3.4, treatment of compound 4.22 with

methylethylketone (CH3CH2COCH3) also generated a UV active (254 nm) component, indicating that the carbonyl (C=O) functional group in ketones and probably aldehydes is susceptible to attack by the ammonium group of compound 4.22. Therefore, we tested the

reactivity of 4.22 with several ketones (e.g, acetophenone, cyclohexanone, benzophenone,

methylethylketone) and aldehydes (e.g, acrolein, acetaldehyde, anisaldehyde and 2,4-

dimethoxybenzaldehyde) in benzene without addition of any catalyst. With the exception

of benzophenone, all of theses ketones and aldehydes presumably generated iminium-

type compounds as demonstrated by UV/TLC inspection at 254 nm. However, carboxylic

acids (e.g, cinnamic acid) treated with 4.22 did not generate iminium-type compounds.

Two iminium compounds (4.24 and 4.25 in Scheme 4.3), derived from cyclohexanone

and 2,4-dimethoxybenzaldehyde, precipitated from benzene and their structures were

confirmed by 1H-, 13C-NMR and HRMS.

The unique chemical reactivity of compound 4.22 may be of great interest to

organic chemists. A plausible mechanism for the reaction of compound 4.22 with ketones

or aldehydes, involving acidic catalysis by an “internal” hydrogen atom, is shown in

Scheme 4.4 using acetone as an example. A free electron pair of the carbonyl oxygen of acetone captures a proton of the ammonium group of 4.22. The protonated carbonyl group is now sufficiently electrophilic to be attacked by the emerged nucleophilic amine group to generate a tetrahedral intermediate. Eventually, the formation of a stable conjugated system between carborane cluster and iminium (C=N+) group in 4.23 is the

driving force for the elimination of water in the last step.

H OH O H H H H H H H N H H H N H N N H H H H H2O O

4.22 Intermediate 4.23

Scheme 4.4. Plausible mechanism of generation of 4.23 from 4.22 with acetone

In general, amines react with carbonyl groups of aldehydes or ketones in the presence of acid catalyst under reflux and formation of imine- or iminium-containing compounds is accomplished by removing water through azeotropic distillation. In contrast, acetone and compound 4.22 do not require any acid catalyst, reflux, and azeotropic distillation to generate compound 4.23. In addition, hydrolysis of 4.23 occurred under very mild condition (5% NaHCO3/EtOH, rt, 2 h) to regenerate 4.22.

Therefore, we believe the reactivity of the ammonium function attached to the

zwitterionic nido m-carboarne cages of the 3CTAs (4.14.B-4.19.B) will be of no

consequence for their biological activities under physiological conditions. However, it is

conceivable that the unique chemical properties of compound 4.22 could be utilized to

facilitate organic reactions (e.g. Michael addition) or to detect carbonyl impurities in

analytical chemistry.

4.3.6. Summary and conclusions

The zwitterionic ammonium-substituted nido m-carborane cluster (4.22) was

synthesized and its experimental data were compared with those calculated at the ab

initio HF and DFT levels using Gaussian 03.194 GIAO/11B-NMR chemical shifts for 82 compound 4.22 obtained at the B3LYP/6-31G* and B3LYP/6-31G** levels showed

+ strong correlations with the experimental values. The zwitterionic NH3 -nido m- carborane 4.22 exhibited unique chemical properties by reacting with the carbonyl group of ketones or aldehydes without addition of acid catalyst at room temperature. The zwitterionic iminium-substituted compound 4.23 showed a bathochromic shift in UV experiment due to the stable conjugation of the σ-aromatic carborane cage with the iminium group. The unique structural, physicochemical, and chemical properties of the

+ novel zwitterionic NH3 -nido m-carborane structure may lead to a wide range of novel applications in organic chemistry and boron chemistry.

4.4. Outlook

Based on the evaluation of its enzymatic and physicochemical properties, zwitterionic compound 4.14.B has been identified as one of two “2nd Generation 3CTA” which have

the potential to replace N5-2OH, the current lead compound of our 3CTA library. The

other “2nd Generation 3CTA” is the compound 4.26 shown in Figure 4.8. Compound 4.26 was synthesized and evaluated as a boron-delivery agent by Narayanasamy et al.211

O H H NH3 O OH N N HO N O O HO N O OH OH O OH 4.14.B 4.26 OH

Figure 4.8. Chemical structures of 2nd generation 3CTAs

83 The nature of chirality in 1,2-disubstituted nido o-carboranes or 1,7-disubstituted

nido m-carboranes has been poorly studied.174,222,223 Therefore, the possibility of simple

chromatographic separation of pure epimers of 4.14.B may be a useful tool to initiate

more detailed studies on the chirality of nido m-carborane. The dThd moiety in both

224 epimers of 4.14.B could be removed with hydrazine (NH2NH2). Alternatively, one

+ could attach dThd or a comparable structure to NH3 -nido m-carborane via a linker that is easily cleaved. To substantiate our hypothesis that pure epimers rather than atropisomers

of 4.14.B have been separated by HPLC, analysis of two pure epimers by X-ray

chrystallography is warranted. X-ray crystal structure of iminium compounds 4.23, 4.24

or 4.25 would also be determined.

84 CHAPTER 5

DISCOVERY OF NOVEL ANTIBIOTICS TARGETING THYMIDINE KINASE

AND THYMIDINE MONOPHOSPHATE KINASE

FROM BACILLUS ANTHRACIS

5.1. Introduction

Bacillus anthracis (B. anthracis), the agent that causes anthrax infections, is a

Gram-positive, facultative, anaerobic bacterium. Anthrax infection is classified into three

types by the route of exposure: cutaneous, inhalational, and gastrointestinal anthrax.225

Humans can be infected with anthrax by handling infected animals, inhaling anthrax spores, or eating uncooked meat of infected animals.225 Anthrax spores can survive for

many years in the soil and can be easily transmitted to humans. Regardless of the

exposure route to humans, anthrax spores are phagocytosed by macrophages where they

germinate.226 Cutaneous anthrax is the most common while the inhalational anthrax is the

most deadly form.

Interest in anthrax pathogenesis emerged as a consequence of the intentional attack via contaminated letters with anthrax spores in 2001 in the USA, and thus, the primary concern with anthrax in the 21st century is its use as a biological weapon.227

FDA-approved antibiotics for the treatment of anthrax infections include ciprofloxacin

85 (fluoroquinolones), doxycycline (tetracyclines), and penicillin G (β-lactams).228,229 An

anthrax vaccine (Biothrax®, BioPort Corporation, Lansing, MI) is available for

prevention of anthrax disease.229 However, resistance could be developed towards the

current antibiotics and vaccination of the large population is difficult. Therefore, there is

an urgent need to discover novel antibiotics for the treatment of unvaccinated individuals

who may become exposed to anthrax or infected with antibiotic-resistant strains of B. anthracis.

5.2. Rationale

The physiological function of typical salvage pathway enzymes in prokaryotes is not known. But it is very likely that they complement the de novo pathway of the DNA precursor synthesis. It is also possible that these enzymes are essential for colonization and pathogenicity in certain situations of bacterial growth. A large fraction of multidrug- resistant Staphylococci has proven to be dependent on an exogenous supply of dThd for growth.230,231 B. anthracis strains lacking thymidylate synthase (TS) activity relied on the

salvage pathway for dTTP production for survival.232 Figure 5.1 depicts biochemical

pathways for the synthesis of dTTP in B. anthracis. B. anthracis thymidine kinase

(BaTK) catalyzes the production of dTMP from dUrd and dThd in the salvage pathway

while TS plays a pivotal role in transferring a methyl group from 5,10-methylene

tetrahydrofolate to dUMP for dTMP production in the de novo pathway. dUMP is

synthesized from dUTP by a dUTPase and from dCMP by a dCMP deaminase in the de

novo pathway. The role of thymidine monophosphate kinase (TMPK) for dTTP

86 production is even more crucial than those of TK and TS because it is supplied with dTMP both by salvage and de novo pathways.

Figure 5.1. Salvage and de novo biochemical pathways for dTTP synthesis

TK: Thymidine kinase, TMPK: Thymidine monophosphate kinase, TS: Thymidylate synthase, DHFR: Dihydrofolate reductase, DHF: Dihydrofolate, THF: Tetrahydrofolate. dUTP: 2´-Deoxyuridine triphosphate, dCMP: 2´-Deoxycytidine monophosphate

BaTK may be an attractive molecular target for novel antibiotics for the treatment of anthrax infections. It could be utilized to activate a nucleoside-based prodrug, which is converted to a toxic dNTP by dNKs in a “traditional” way36,41,42 or it could be targeted

87 with a BaTK-specific inhibitor. In the latter case, a BaTK-specific inhibitor may be used in combination with an inhibitor of TS or other crucial enzymes in the de novo pathway.

To our knowledge, substrates or inhibitors targeting bacterial TK have not yet been developed for therapeutic purposes. Recently, however, the known TK substrate 1-(2´- deoxy-2´-fluoro-β-D-arabinofuranosyl)-5-[125I]iodouracil (F[125I]IAU) proved to be a

useful tool for the diagnosis of various localized bacterial infections by Staphylococcus

aureus and Enterococcus faecalis.54

B. anthracis thymidine monophosphate kinase (BaTMPK) is a more attractive

target than BaTK because dTMPs supplied by salvage and de novo pathways are

diphosphorylated only by BaTMPK. There is no report on transport of dTDP into bacteria

and, therefore, it is likely that dTDP production in B. anthracis relies solely on the action

of BaTMPK. Unless B. anthracis possesses an unknown alternative mechanism to

generate or transport dTDP, BaTMPK provides an excellent molecular target for the

discovery of novel antibiotics for the treatment of anthrax infections.

5.3. Evaluation of agents with established molecular targets in mammalian and viral systems as potential antibiotics against B. anthracis

The structures of agents used in enzyme and toxicity studies described below are shown in Figure 5.2. Their established biological activities in mammalian and viral systems are summarized briefly as follows. The dUrd analogues substituted with halogen atoms or the ethyl group at the 5-position (5-FdUrd, 5-CldUrd, 5-BrdUrd, 5-IdUrd, and

5-EtdUrd) are known to be good substrates for hTK1 and their triphosphates are

substrates for human DNA polymerase. Floxuridine (5-FdUrd) and 5-FU are strong TS

88 inhibitors. dThd analogues substituted with bulky groups at the N-3 position (3-

isopropyl-dThd, 3-propargyl-dThd, and N5-2OH) are effectively phosphorylated by

hTK1 and UuTK, however, not much is known about their subsequent metabolism.12,43 dThd analogues modified at the 3´-position are tolerated moderately by hTK1 as substrates.147,233 They are also further phosphorylated to their triphosphate forms, which

are relatively poor substrates of human DNA polymerase. However, triphosphates of

AZT and d4T are good HIV (human immunodeficiency virus) reverse transcriptase

inhibitors.234

The anticancer agents gemcitabine, ara-C, and cladribine are all effectively

monophosphorylated by dCK. Their corresponding triphosphates are powerful

competitive inhibitors of human DNA polymerase and they terminate DNA synthesis

through their incorporation into DNA. Gemcitabine is also a highly effective inhibitor of

RR, another key enzyme in the de novo pathway of DNA synthesis. Acyclovir,

ganciclovir, and BVDU are converted to their monophosphates by HSV-TK but not by

hTK1. Their triphosphates are inhibitors of HSV-DNA polymerase. Ganciclovir is also

monophosphorylated by cytomegalovirus (CMV) pUL97 protein kinase and its

triphosphate is an effective inhibitor of DNA polymerase, which is utilized in HSV-

tk/ganciclovir suicide gene therapy. Hydroxyurea is a RR inhibitor and 6-mercaptopurine

(6-MP) inhibits various enzymes specific to purine nucleotide de novo biosynthesis.

O O O R R NH N NH

N O N O N O HO HO HO O O O

OH OH R AZT: R = N3 5-FdUrd: R = F FLT: R = F 5-EtdUrd: R = Et 3-Methyl-dThd: R = CH3 3'-Azidomethyl-dThd: R= CH2N3 5-CldUrd: R = Cl 3-Propargyl-dThd: R = CH2CCH 3'-Fluoromethyl-dThd: R = CH2F 5-BrdUrd: R = Br 3-Isopropyl-dThd: R = CH(CH3)2 5-IdUrd: R = I 3'-Amino-dThd: R = NH2 3'-Aminomethyl-dThd: R = CH2NH2 3'-Hydroxymethyl-dThd: R = CH2OH Br O O O I O O NH NH NH NH NH N O N O N O HO OH HO N O N O O O O HO HO F F O O OH OH OH O BVDU L-FMAU D-FIAU d4T DOT

NH2 NH2 NH2 SH N N N N N N N O N O N N Cl N N HO HO HO H O O O HO F 6-Mercaptopurine OH OH F OH

Ara-C Gemcitibine Cladrabine

O O O N O NH F N NH NH OH N N NH H2N N HO 2 N N NH N O H HO 2 O H O 5-FU Hydroxyurea OH Acyclovir Ganciclovir

Figure 5.2. Chemical structures of the existing compound library

90 PRa (%) PRa (%) IC at 6 h IC at 20 h Compound 50 50 BaTK hTK1 (µM) (µM) 5-FdUrd (Floxuridine) 91 ± 5 9512 7.0 1.8

5-FU - - 2.9 2.9

5-EtdUrd 83 ± 6 8012 - -

5-CldUrd 80 ± 4 19612 > 500 > 500

5-BrdUrd 67 ± 4 80235 > 500 > 500

5-IdUrd 88 ± 5 17012 - -

BVDU < 0.1 < 1235 > 250 > 250

3-Methyl-dThd 35 ± 6 4312 - -

3-Propargyl-dThd 31 ± 3 2112 > 500 > 500

3-Isopropyl-dThd 23 ± 6 1712 > 200 > 200

N5-2OH 30 ± 5 41235 10.0 22.1

AZT 47 ± 8 5212 > 1000 > 1000

d4T 8 ± 1 743 > 500 > 500

FLT 68 ± 3 3012 > 500 127

D-FIAU 79 ± 6 7612 - -

L-FMAUb - - > 500 > 500

DOTb - - > 500 > 500

3´-Azidomethyl-dThd 31 ± 7 1512 - -

Table 5.1. PRs and in vitro growth inhibition of the existing compound library

Table 5.1 continued

91 Table 5.1 continued

3´-Fluoromethyl-dThd 24 ± 4 1512 - -

3´-Amino-dThd 25 ± 2 - - -

3´-Aminomethyl-dThdc - - > 1000 > 1000

3´-Hydroxymethyl-dThdc - - > 1000 > 1000

Gemcitabine - - 0.34 0.11

Ara-C - - >1000 >1000

Cladribine - - >250 >250

Acyclovir - - >1000 >1000

Ganciclovir - - >500 >500

Hydroxyurea - - >500 >500

6-Mercaptopurine - - >200 >200

Table 5.1. PRs and in vitro growth inhibition of the existing compound library. a PR for dThd was set to 100 with both TKs. The concentrations of compounds and [γ-32P]ATP were 100 µM. b Compounds were a gift from Dr. Serge Van Calenbergh from the Ghent University, Ghent, Belgium. c Compounds were a gift from Dr David Chu from the University of Georgia, Athens, GA. Abbreviations: 5-FdUrd: 5-Fluoro-2´-deoxyuridine, 5-FU: 5-Fluorouracil, 5-EtdUrd: 5-Ethyl-2´- deoxyuridine, 5-CldUrd: 5-Chloro-2´-deoxyuridine, 5-BrdUrd: 5-Bromo-2´-deoxyuridine, 5-IdUrd: 5-Iodo- 2´-deoxyuridine, BVDU: 5-Bromovinyl-2´-deoxyuridine, 3-Methyl-dThd: 3-methylthymidine, 3- Propargyl-dThd: 3-(2-propyn-1-yl)thymidine, 3-Isopropyl-dThd: 3-Isopropylthymidine, N5-2OH: 3-[5-(2- (2,3-Dihydroxyprop-1-yl)-o-carboran-1-yl)pentan-1-yl]thymidine, AZT: 3´-Azido-3´-deoxythymidine, d4T: 2´, 3´-Didehydro-3´-deoxythymidine, FLT: 3´-Fluoro-3´-deoxythymidine, D-FIAU: 2´-Deoxy-2´- flouro-5-iodo-D-arabinofuranosyluracil, L-FMAU: 2´-Deoxy-2´-flouro-5-methyl-L-arabinofuranosyluracil, DOT: 2-Hydroxymethyl-1,3-dioxolan-4-ylthymine, 3´-Azidomethyl-dThd: 3´-Azidomethyl-3´- deoxythymidine, 3´-Fluoromethyl-dThd: 3´-Fluoromethyl-3´-deoxythymidine, 3´-Amino-dThd: 3´-Amino- 3´-deoxythymidine, 3´-Aminomethyl-dThd: 3´-Aminomethyl-3´-deoxythymidine, 3´-Hydroxymethyl- dThd: 3´-Hydroxymethyl-3´-deoxythymidine, Gemcitabine: 2´-Deoxy-2´,2´-difluorocytidine, Ara-C: Arabinosylcytosine, Cladribine: 2-Chloro-2´-deoxyadenosine, Acyclovir: 9-[(2-Hydroxyethoxy)methyl] guanine, Ganciclovir: 9-{[2-Hydroxy-1-(hydroxymethyl)ethoxy]methyl}guanine, 6-MP: 6-Mercaptopurine.

92 5.3.1. BaTK and hTK1 PRs

The BaTK PRs of compounds substituted with halogens or the ethyl group at the

5-position (5-F, 5-Cl, 5-Br, 5-I, and 5-EtdUrd) ranged from 67 to 91% relative to that of

dThd (Table 5.1). The activity decreased drastically (< 0.1%) with larger substitution,

such as the bromovinyl group in BVDU. All dThd analogues substituted at the 3´-

position were good substrates of BaTK (24-68% PRs) except for d4T. Moderate activities

(23-35% PRs) were also observed for dThd analogues modified at the N-3 position. D-

FIAU had a high relative PR of 79% in BaTK, which supports its potential use as a diagnostic agent for localized bacterial infections.54 Overall, the PRs observed for BaTK

were comparable with those observed for hTK1 (Table 5.1). Except AZT, however, the

BaTK PRs for most of the tested 3´-substituted compounds were higher than those for

hTK1, indicating that selectivity for BaTK versus hTK1 may be achieved through 3´- modification. The PTAs described in this chapter were carried out in collaboration with

Dr. Staffan Eriksson and coworkers.

5.3.2. Molecular modeling

Extensive studies with M. tuberculosis TMPK indicated sensitivity to inhibition

by dThd and dTMP analogues with 3´-modifications such as the 3´-azido-, 3´-fluoro,

3´-azidomethyl and 3´-fluoromethyl group.236-238 As shown in Table 5.1, the PRs of 3´-

azidomethyl-Thd, 3´-fluoromethyl-Thd, and FLT were about two times higher with

BaTK compared with hTK1. In order to elucidate whether 3´-modifications at dThd have the potential to generate selective inhibitor/substrate for BaTK versus hTK1, we investigated the dimension of the binding pocket around the 3´-edge of the dThd scaffold

93 in a homology model of BaTK and the crystal structure of hTK1 using Site ID module

implemented in Sybyl 7.1 (Figure 5.3).

Figure 5.3. Dimensions of the dTTP binding pocket in hTK1 crystal structure (A) and the BaTK homology model (B).

Although there is higher sequence identity (62%) between BaTK and CaTK than between BaTK and hTK1 (40%) or UuTK (43%), crystal structures of the latter two were used as templates to build the homology model of BaTK. These two TKs presumably represent ‘closed’ form while CaTK may represent an ‘open’ form as discussed in

Chapters 1 and 3. Standard docking programs are able to enrich known ligands for a specific protein target out of large libraries consisting of ligands and non-ligands.202,239

However, the nature of the protein structure plays a crucial role in the level of enrichment.

In general, holo (closed) conformations appear to be more effective than apo (open) conformations.202 We hypothesize that, in particular, for the identification of inhibitor

94 binding, holo conformations are essential. As already discussed in Chapter 3, the computational analysis of substrate binding may also necessitate the use of apo conformations.

Comparative Site ID analysis of the active site of BaTK homology model and hTK1 crystal structure showed that BaTK has a larger cleft around the C-3´/C-4´ edges of

2´-deoxyribose than hTK1 (Figure 5.3), indicating that modification at this position may result in selective binding to BaTK versus hTK1. Indeed, based on both the docked poses and their binding energies, several of C-3´-branched dThd triphosphates analogues (e.g., -

CH2C≡CH, -CH2CH=CH2, -CH2N3, -CH2SH, -NHCH3, -OCH3 and -SCH3) built in silico

showed better binding to the BaTK homology model than to the hTK1 crystal structure

(PDB ID: 1W4R) when docked with the FlexX module of Sybyl 6.9. However, in vitro

toxicity studies of AZT, FLT, 3´-aminomethyl-dThd, and 3´-hydroxymethyl-dThd in B.

anthracis Sterne indicated that 3´-modification at dThd alone may not have the potential

to exert significant toxicity in B. anthracis (Table 5.1).

5.3.3. In vitro toxicity studie with B. anthracis Sterne

B. anthracis Sterne strain (34F2), an attenuated strain of B. anthracis, was used in

in vitro toxicity studies. It has been used previously as a surrogate for the B. anthracis

Ames, a virulent strain.240 We carried out in vitro toxicity studies of the existing library

with B. anthracis Sterne. Among all screened dThd and dUrd analogues, only

Floxuridine (1.8 µM), N5-2OH (22.1 µM) and FLT (127 µM) had IC50 values < 200 µM

at 20 h incubation. Gemcitabine and 5-FU showed low IC50 values at 0.11 µM and 2.9

µM, respectively. This result implied that the functions of RR or TS are crucial for 95 growth of B. anthracis Sterne. Floxuridine is a very good substrate both of BaTK (PR:

91%) and hTK1 (PR: 95%). This agent is an effective inhibitor of TS as a monophosphate, and a good terminator of DNA biosynthesis by incorporating into both

DNA and RNA in triphosphate form. The moderate toxicity observed for N5-2OH appeared to be due to its high lipophilicity (see Chapter 3) rather than its capacity to function as a BaTK substrate and/or inhibitor. Its high lipophilicity may allow N5-2OH to interact with membrane components of B. anthracis Sterne, thereby causing toxicity.70,181

Lipophilicity-dependent toxicity of carborane-containing compounds has been previously demonstrated in mammalian systems.130,133 FLT is phosphorylated more than twice as

effectively by BaTK than by hTK1 and it also showed moderate toxicity in B. anthracis

Sterne after 20 h incubation. FLT toxicity was not observed after 6 h incubation,

indicating that toxic FLT metabolites are accumulated in B. anthracis Sterne over a

longer time period. Except for BVDU, all other dUrd analogues were efficiently

phosphorylated by BaTK in PTAs but they were apparently not toxic to B. anthracis

Sterne in in vitro toxicity studies.

This is in a sharp contrast with mammalian or viral systems which are highly sensitive to these compounds. Possible explanations for the lack of toxicity could be the

fact that these agents are not effectively transported into the interior of B. anthracis

Sterne or the conversion of the 5´-monophosphates to triphosphate forms by TMPK and

NDPK of B. anthracis Sterne is hampered. It is also conceivable that the triphosphates

are not substrates of B. anthracis Sterne DNA polymerase or they are ineffective as

feedback inhibitors of BaTK.

96 However, high and moderate toxicities observed for Floxuridine and FLT indicate

that transport of dThd analogues with 3´- and 5-modifications into B. anthracis Sterne is

possible and that their metabolites can exert toxicity. Some reports indicate low utilization levels for TKs in Gram-positive bacteria and thus low dependence on salvage pathways for their DNA synthesis.241,242 This may, in particular, be the case for the M9

minimal growth medium used in our toxicity studies, which only contains phosphate, chloride, glucose, and amino acids but no nucleoside or nucleobase supplements. This may cause downregulation of nucleoside transport mechanism and salvage pathway enzymes in B. anthracis Sterne. This explanation would also account for the high

toxicities observed for floxuridine and 5-FU, which are TS inhibitors. Remarkable is the observed toxicity of gemcitabine in B. anthracis Sterne, which is comparable to that of enrofloxacin, the fluoroquinolone-class antibiotic we used as a positive control for in vitro toxicity studies. Gemcitabine utilizes both salvage (dCK) and de novo pathway enzymes (RR) for its activity. This substantiates the initial assumption (Chapter 5.2) that

both pathways must be targeted in B. anthracis for nucleoside-based antibiotic therapy of

anthrax. It remains to be determined whether gemcitabine can be effective as an antibiotic

at plasma concentrations that are significantly lower than those used in cancer

chemotherapy.

The in vitro toxicity studies described in Chapter 5.3.2 were carried out in

collaboration with Dr. Andrew J. Phipps in the OSU Department of Veterinary

Biosciences.

5.4. BaTK- and/or BaTMPK-specific inhibitors

97 5.4.1. Drug design

Studies described in Chapter 5.3 indicated that the concept of dThd or dUrd

prodrug-based antibiotics for anthrax may have limited potential. Therefore, we focused

our efforts on the discovery of agents that could function as selective inhibitors of BaTK

and/or BaTMPK that do not require metabolic activation. Such inhibitors could compete with endogenous substrates (dThd, dUrd, dTMP) of BaTK/BaTMPK and thereby

decrease dTTP production. The resulting imbalanced dNTP pool could affect DNA

synthesis in B. anthracis, and thus, reduce its growth and spread.

BaTK belongs to the TK1-like kinase and has 40% and 43% sequence identity with hTK1 and UuTK, respectively. Crystal structures of hTK1 and UuTK were

determined very recently,12,20,23 most of which are in complex with dTTP, the

endogenous feedback inhibitor of hTK1 and UuTK, in the active site. Therefore, dTTP was utilized as a template to design novel BaTK/BaTMPK inhibitors.

Compounds containing a triphosphate group, however, are inherently unstable

under physiological conditions and they can not cross cell membranes because of their

highly ionic character. These features render a triphosphate-containing compound

completely unsuitable as a drug. Therefore, research on stable phosphate mimics with

increased lipophilicity has been extensive.243-275 These mimics include acyclic structures

and five- or six-membered homo- or heterocyclic (N, O, S) ring systems substituted with

e.g. hydroxyl, amino, methoxy, sulfonate, sulfonamide, carboxylic, keto, thioketo, chlorine, fluorine, urea, thiourea, phosphoamide, or carbamate groups as well as

triphosphates in which backbone oxygen atoms were replaced with e.g. amino, methylene,

fluoromethylene, hydroxymethylene, chloromethylene, borane, and cyanoborane groups.

NH2 NH2 N N N N O O O N N H N N H2N S O HO N S O O O O O F OH OH OH OH

Nucleocidin Salicyl-AMS

Figure 5.4. Known antibiotics substituted with phosphate mimics at the 5´-position

Some of these phosphate mimics had already found successful application in the

(bio)synthesis of natural and synthetic antibiotics.245,246,248-251,256-260,269,272 These include

the natural product Nucleocidin (Figure 5.4), an inhibitor of protein synthesis with broad

antibacterial spectrum,245,250,258,272 and Salicyl-AMS (Figure 5.4), an inhibitor of the

siderophore biosynthesis with an salicylsulfamoyl group at the 5´-position.246,248 Both of these agents demonstrated antimicrobial activity, and therefore, it is reasonable to assume that BaTK/BaTMPK inhibitor containing stable lipophilic phosphate mimics at the 5´- position should be able to enter the interior of B. anthracis.

Despite significant structural differences (Figures 5.6 and 5.7), TKs and TMPKs catalyze very similar biochemical processes. This may be the reason why TKs of most

HSVs have both TK and TMPK activity.276,277 Both enzymes possess a binding site for

dThd or dTMP, embedded in the interior of the enzymes, an adenosine binding site,

located on the surface of the enzymes, and a bridging phosphate binding site, which

accommodates either three (TK) or four (TMPK) phosphate moieties. Therefore, design

99 strategies for BaTK inhibitors based on phosphate mimics are also applicable to

BaTMPK inhibitors.

The design of selective inhibitors of BaTMPK may be more attractive than those

of BaTK because the sequence identity between human TMPK (hTMPK) and BaTMPK is only 21% while that of hTK1 and BaTK is 40%. Various crystal structures of hTMPK

as well as those of Escherichia coli (E. coli), Mycobacterium tuberculosis (M.

tuberculosis), Yeast, and Staphylococcus aureus (S. aureus) have been determined.278-285

In particular, E. coli and S. aureus TMPK (SaTMPK) have 34% and 49% sequence identity with BaTMPK, which allowed us to build a homology model of BaTMPK.

O O O N O O O HO P O P O P O NH NH OH OH OH O R N O O N O OH R H N S N a e dTTP N N O O O b d c OH OH Sulfonamide-based dThds Triazole-based dThds

O O O

NH NH NH

N O N O R O N O H H H H H N N N N N O O R O R O S OH OH OH Urea-based dThds Thiourea-based dThds Amide-based dThds

Figure 5.5. Design of phosphate-mimicking dThd analogues as BaTK/BaTMPK inhibitors

100 Figure 5.5 shows strategies for the design of BaTK and BaTMPK inhibitors

containing phosphate mimics. To the best of our knowledge, none of these phosphate

mimics has been used previously for the design and synthesis of dThd analogues. The

dThd scaffold was kept intact because it provides the element of selective affinity for

both enzymes. Sulfonamide-, urea-, thiourea-, amide-, and triazole linkers were chosen to

replace the α-phosphate moiety of dTTP. In particular, triazole has an interesting linker

property because it has a much stronger dipole moment than an amide bond, and thus, the

nitrogen atoms of the triazole group act as hydrogen bond acceptors.286 To increase

lipophilicity, the benzene moiety was attached as a replacement of the β-phosphate of

dTTP. Additional polar groups (e.g. SO2CH3, CO2CH3, and SO2NH2) were attached to

the benzene moiety to substitute for the terminal γ-phosphate moiety of dTTP. All three groups were expected to interact with the phosphate-loop (P-loop) region of both enzymes via hydrogen-bonding and electrostatic interaction.

5.4.2. Synthesis

O O O O

NH NH NH NH

N O N O N O N O a b c HO TsO N3 H2N O O O O

OH OH OH OH

dThd 5.1 5.2 5.3

Scheme 5.1 Synthesis of 5´-amino-5´-deoxythymidine as a key intermediate

Reagents and conditions: (a) p-toluenesufonyl chloride (1.1 eq), pyridine, 0 °C Æ rt; (b) NaN3, DMF, 70 °C, 12 h; (c) H2, 5% Pd/C, EtOH, RT, 24 h.

101 The preparation of the designed compounds shown in Figure 5.5 was achieved by

utilizing compounds 5.2 and 5.3 as key intermediates. Compound 5.3 was obtained

according to a reported procedure287,288 by selective tosylation of the 5´-position of dThd, followed by the introduction of azide, and palladium-catalyzed hydrogenation.

5.4: R= SO2CH3

O O SO2CH3

NH NH 5.5: R= N O O N O a H SO CH H2N R S N 2 3 O O O 5.6: R= OH OH

5.3 5.7: R=

Scheme 5.2. Synthesis of sulfonamide-linked dThd inhibitors

Reagents and condition: (a) the appropriate sulfonyl chloride, TEA, DMF, rt, 6 h.

Sulfonamide-linked dThd inhibitors (5.4-5.7) were prepared in 65-80% yield by reacting 5.3 with the appropriate sulfonyl chlorides [4-(methylsulfonyl)benznesufonyl chloride for 5.4, 3-(methylsulfonyl)benzenesulfonyl chloride for 5.5, 2-

(methylsulfonyl)benzenesulfonyl chloride for 5.6, 1-naphthalenesulfonyl chloride for 5.7] in the presence of triethylamine (Scheme 5.2).

102 O O NH NH N O H H N O N N H N a O 2 CH3S O S OH OH 5.8: ortho 5.3 5.9: meta a 5.10: para

O O

NH NH

N O O N O H H H H N N N N O O CH S O 3 O O OH OH 5.13 5.11: meta 5.12: para

Scheme 5.3. Synthesis of thiourea- and urea-linked dThd inhibitors

Reagents and condition: (a) the appropriate phenylisothiocyanate or phenylisocyanate, DMAP, pyridine, rt, 12 h.

Thiourea-linked compound 5.8 was prepared by reacting compound 5.3 with 2-

(methythio) phenylisothiocyanate and catalytic amount of DMAP in pyridine. The meta- and para-isomers (5.9-5.10) were prepared in 73% and 83% yield by using 3-

(methylthio)- and 4-(methylthio) phenylisothiocyanate, respectively. Identical reaction conditions were used for the synthesis of the urea-linked dThd inhibitors 5.11-5.13.

Treatment of 3-(methylthio)-, 4-(methylthio)phenylisocyanate, and methyl 3- isocyanatobenzoate with 5.3 afforded 5.11 (68% yield), 5.12 (73% yield), and 5.13 (52% yield), respectively.

103 O O 5.14: R= SO NH NH 2 2 NH N O H N N O 2 a H O R N 5.15: R= SO2CH3 O OH O OH 5.3 5.16: R= SCH3 a O O NH 5.17: R= CO2CH3 O NH O N O OMe N O N H CO2CH3 N O O O 5.18: R= O OH OH 5.19.I 5.19

Scheme 5.4. Synthesis of amide-linked dThd inhibitors

Reagents and condition: (a) the appropriate benzoic acid, DCC, HOBt, DMF, rt, 12 h.

Various benzene carboxylic acids (see experimental section) were coupled with

5.3 using 1-hydroxybenzotriazole (HOBt) and 1,3-dicyclohexylcarbodiimide (DCC) to afford the amide-linked inhibitors 5.14-5.18 in yields ranging from 38% to 58%. When methyl hydrogen phthalate was reacted with 5.3, compound 5.19 was synthesized in 28% yield via the intermediate 5.19.I, as shown in Scheme 5.4. This reaction was presumably facilitated by the close proximity of CO2Me and NH groups resulting in the formation of the phthalimide via intramolecular cyclization.

104 5.20: R=

O O

NH NH 5.21: R= F R N O N O a N N N 3 N O O 5.22: R= OCH3

OH OH N 5.2 5.23: R=

Scheme 5.5. Synthesis of triazole-linked dThd inhibitors

Reagents and condition: the appropriate alkynes, sodium ascorbate, CuSO4, H2O/t-BuOH (1:1), vigorous stirring, rt, 48 h.

The triazole-linked inhibitors 5.20-5.23 were prepared by reacting the azide 5.2

with the appropriate alkynes (see experimental section for details). Formation of 1.4-

substituted-1,2,3-triazoles from azides and alkynes had been described as ‘click

chemistry’.286 No protecting groups are required in this reaction. Copper (I) is the most

powerful catalyst of this 1,3-dipolar cycloaddition of azides and alkynes.289 We used the

‘copper (II)/ascorbate system’, which generates the Cu (I) catalyst in situ by reducing

290 CuSO4 with sodium ascorbate. To our knowledge, this is the first report on the triazole-

linked dThds. However, the obtained yields were very low (8-15%).

5.4.3 Biological evaluation

In vitro toxicity studies of compounds 5.1-5.23 were carried out using B.

anthracis Sterne. As shown in Table 5.2, all of the thiourea- and urea-linked compounds

except compound 5.13 produced inhibition at 6 h incubation. In particular, the IC50 values 105 of compounds substituted with SCH3 group at the meta position of the phenyl ring (5.9 and 5.11) were very low with 5.3 µM and 0.48 µM, respectively. However, their activities deteriorated dramatically at 20 h incubation. The amide-linked compounds with

SCH3 (5.16) and CO2Me (5.17) at phenyl ring also displayed significant inhibitory activity at 6 h but not at 20 h. Compounds 5.9-5.12 and 5.16-5.17 have common structural features, which could have been metabolized by enzymes of B. anthracis

Sterne. SCH3 is easily oxidized to sulfoxide or sulfone and CO2Me could be hydrolyzed by esterases to give CO2H. Urea and thiourea bonds could also be cleaved to produce the

inactive precursor 5.3. This hypothesis is supported by the IC50 of compound 5.15, a

potential metabolite of compound 5.16, which was inactive at 6 h and 20 h. Also, we

observed decomposition of urea-, thiourea-, and amide-linked dThds in DMSO even if

they were kept at -20 °C.

The structurally different triazole-linked dThd analogues were active at 6 h in the

range of 84 to 430 µM concentration. Interestingly, compound 5.21 retained its activity to some degree until 20 h (IC50: 303 µM). It is conceivable that the triazole-linked

compounds are in general more resistant to metabolism or hydrolysis compared with the

urea-, thiourea-, and amide-linked compounds and thus, displayed activities up to 20 h.

While transformation to inactive metabolites may be an explanation for the deteriorating

activities observed for several compounds in B. anthracis Sterne, it could be another

explanation that they have a bacteriostatic rather than a bactericidal effect and delayed

the growth of B. anthracis Sterne, which eventually reached a plateau of growth after 20

106

In vitro growth inhibition of B. anthracis Sterne Compound IC50 (µM) at 6 h IC50 (µM) at 20 h 5.1 > 1000 > 1000 5.2 > 1000 > 1000 5.3 > 1000 > 1000 5.4 > 1000 > 1000 5.5 > 1000 > 1000 5.6 > 1000 > 1000 5.7 > 1000 > 1000 5.8 161 > 1000 5.9 5.3 > 1000 5.10 309 > 1000 5.11 0.48 > 1000 5.12 16.5 > 1000 5.13 > 1000 > 1000 5.14 > 1000 > 1000 5.15 > 1000 > 1000 5.16 6.7 > 1000 5.17 19.1 > 1000 5.18 > 1000 > 1000 5.19 > 1000 > 1000 5.20 328 > 1000 5.21 84 303 5.22 430 > 1000 5.23 N/A N/A

Table 5.2. In vitro IC50 of dThd analogues against B. anthracis Sterne

N/A: not determined

107 5.4.4. Molecular modeling

5.4.4.1. B. anthracis thymidine kinase (BaTK)

(A) (B) (C)

Figure 5.6. The hTK1 crystal structure with dTTP (A) and the BaTK homology model docked with compound 5.11 (B) and 5.21 (C)

Docking of compounds 5.1-5.23 into the crystal structure of hTK1 and the

homology model of BaTK (Chapter 5.3.2) demonstrated that all thiourea- and urea-linked

dThds (5.8-5.13) bind to the active site of both enzymes in a fashion that is similar to that

of dTTP (Figure 5.6). The structures of urea-linked 5.11 and triazole-linked 5.21 docked into the active site of the BaTK homology model are shown in Figure 5.5. Both the urea group in 5.11 and the triazole moiety in 5.21 overlapped with the α-phosphate in dTTP, as predicted. Most of the sulfonamide-, amide-, and triazole-linked inhibitors (5.4-5.7,

5.14-5.18, and 5.20-5.23) also showed similar binding patterns compared with that of

108 dTTP in both evaluated 3D-structures. Only the phthalimide-linked compound 5.19 did

not dock to either of these structures.

5.4.4.2. B. anthracis thymidine monophosphate kinase (BaTMPK)

TMPK from S. aureus (SaTMPK) was used as a template to build a homology model of BaTMPK because S. aureus and B. anthracis are Gram-positive bacteria and their TMPKs have high sequence identity (49%) as mentioned in Chapter 5.4.1. Recently, three crystal structures of SaTMPK (PDB ID: 2CCG, 2CCJ, and 2CCK) were determined.291 Surprisingly, two poses of dTMP were found in the one of the SaTMPK

crystal structures (PDB ID: 2CCJ) as shown in Figure 5.7.B. In one dTMP structure, the

monophosphate group interacting with P-loop region was projected towards the putative

ADP binding site (Figure 5.7.B). However, the monophosphate group of the other dTMP

projected towards a pocket generated by the c- and d-loops (Figure 5.7.B).

The guanidine side chain of Arg 106 in the d-loop of SaTMPK interacts with the

α-phosphate of the second dTMP structure and may contribute the dTMP binding. This

arginine residue in the d-loop region is found in SaTMPK, BaTMPK and E. coli TMPK,

but not human, yeast, and M. tuberculosis TMPK. Docking studies of compounds 5.11

and 5.21 into the BaTMPK homology model exhibited that the phosphate-mimicking

portions of their docked poses are located very close to d-loop region, indicating that

these two compounds in BaTMPK may have a binding mode similar to the second dTMP

in SaTMPK (Figure 5.7.C and 5.7.D).

109

(A) (B)

Figure 5.7. (A) Crystal structure of hTMPK with dTMP and ADP, (B) Crystal structure of SaTMPK with dTMP, (C) BaTMPK homology model with 5.11, (D) BaTMPK homology model with 5.21. a: P-loop, b: Lid-region, c: Loop missing in 2CCK, d: Loop with Arg 105 110

(E) (F)

Figure 5.7. (E) BaTMPK homology model with 5.9, (F) Crystal structure of hTMPK with 5.11

The carbonyl oxygen of urea in 5.11 is 1.89 Å away from the guanidine group in

Arg 109 of BaTMPK which corresponds to Arg 106 in SaTMPK. The binding pattern of

compound 5.9 in BaTMPK was similar to the first dTMP binding pose in SaTMPK

(Figure 5.7.E). In contrast, docking of 5.9, 5.11, and 5.21 into the crystal structure

hTMPK crystal structure showed the different binding pattern. The thymine regions of all

three compounds overlapped the β-phosphate of ADP and the phenyl ring in these structures overlapped the 2´-deoxyribose moiety of dTMP substrate (Figure 5.7.F). Such

an inversed binding pattern also has the potential to inhibit substrate binding. Indeed, a

similar inversed binding pattern has been found for AZTMP both in the crystal structures

of hTMPK and of TMPK from M. tuberculosis.189 It has been suggested that this specific

interaction of AZT-MP with these enzymes may contribute to its toxicity in both systems. 111

5.5. Summary and conclusions

A compound library composed of known antiviral and anticancer drugs was

screened to find a lead compound for the development of potential nucleoside-based

antibiotics for the treatment of anthrax infections. PTAs showed that most of dThd and

dUrd analogues of this compound library were good substrates for BaTK as well as hTK1.

Compounds substituted with a bulky group at 3´-position were better substrates of BaTK

than of hTK1, implying this position can be utilized to achieve selectivity for BaTK

versus hTK1. This finding was substantiated by computational Site ID studies. In vitro

toxicity studies of this library in B. anthracis Sterne showed that all of dThd and dUrd

analogues were inactive except FLT and Floxuridine. However, the IC50 of FLT is too

high (127 µM) for selecting it as a lead compound and the activity of Floxuridine appeared to be derived from inhibiting TS rather than from blocking salvage pathway enzymes.

Twenty dThd analogues were designed and synthesized as potential inhibitors of

BaTK and/or BaTMPK. These structures were designed to mimic dTTP, the endogenous feedback inhibitor of BaTK. The phosphate moiety of dTTP was replaced with sulfonamide-, amide-, urea-, thiourea-, or triazole groups in these inhibitors. Some of the urea-, thiourea-, and triazole-linked inhibitors exhibited relatively low IC50 values in

toxicity studies with B. anthracis Sterne, indicating that they may have potential as lead

compounds. Molecular modeling studies of the active compounds revealed that their

binding patterns in BaTK and BaTMPK are similar to those of dTTP in hTK1 and dTMP

in SaTMPK. Overall, a concept of novel antibiotics targeting BaTK and BaTMPK was

112 validated and two compounds (5.11 and 5.21) were selected as lead compounds for

further structural optimization of this series.

5.6. Outlook

The biological evaluation of the twenty potential BaTK and/or BaTMPK

inhibitors should be substantiated with enzyme inhibition studies including hTK1, BaTK,

hTMPK and BaTMPK. Also, crystal structures of BaTK and BaTMPK have to be

generated to replace the homology models of both enzymes used in our studies for the

development of more accurate binding models of active compounds. Finally, further

toxicity studies with active inhibitors, including additional time points between 6 and 20

h, have to be carried out to elucidate the observed phenomenon of deteriorating toxicities

at 20 h. A final analysis of this study will be only possible when these additional experiments are accomplished.

113 CHAPTER 6

SUMMARY AND CONCLUSIONS

TK is a key nucleoside metabolizing enzyme in the DNA biosynthesis in many organisms. It is a promising molecular target for the development of anticancer drugs because it has a very high activity in all types of tumor cells but not in most normal resting cells. Also, the frequent occurrence of TK activity in B. anthracis makes the enzyme a promising target for novel alternative antibiotics for anthrax infections. This dissertation aimed at two research goals: One was to develop hTK1-targeting BNCT agents to treat human GBM and the other was to discover novel BaTK/BaTMPK- targeting antibiotics for anthrax infections.

GBM is one of the most malignant and aggressive of all tumors. Despite significant advances in the treatment of many other cancer types using surgery, chemotherapy, and radiation therapy, the survival of patients diagnosed with GBM has remained almost unchanged for decades. B. anthracis, the agent that causes anthrax, is a

Gram-positive, spore-forming, anaerobic bacterium. Due to the infectiousness of B. anthracis spores by the respiratory route and the high mortality of inhalational anthrax, it can be utilized as a bioweapon. Therefore, both GBM and anthrax infections are major public health concerns in USA.

114 In our research related to the development of novel anticancer agents for the

treatment of GBM, BNCT was chosen as the therapeutic modality. BNCT selectively

kills tumor cells without damaging normal brain tissue by utilizing a nuclear reaction of

boron-10 isotope (10B) with thermal neutrons. Previous SAR studies of 3CTAs identified one promising boron-delivery agent designated N5-2OH. Two feasible synthetic routes for the preparation of 10B-enriched N5-2OH were developed from commercial sources, o-

carborane and decaborane. 10B-enriched N5-2OH was evaluated as a boron-delivery

agent for BNCT in pilot neutron irradiation experiments at MIT. Treatment of rats,

bearing intracerebral F98 glioma, with 10B-enriched N5-2OH and neutron irradiation

resulted in prolonged survival compared with control groups, indicating that 10B-enriched

N5-2OH has a significant potential as a BNCT agent for the treatment of human GBM.

Stereochemical- and geometrical N5-2OH isomers were synthesized and their substrate characteristics for hTK1 were evaluated to complete the SAR studies of the N5-

2OH series. Stereochemical- and geometrical N5-2OH isomers had hTK1 PRs in the

range of 32% to 45% and did not show any significant difference among the isomers.

Binding modes of several geometrical N5-2OH isomers to the active site of hTK1 were

elucidated by carrying out the docking studies of N5-2OH series with the homology

model of hTK1 and the hTK1 crystal structure. The results showed that the bulky

carborane cluster at the N-3 position of dThd projected towards the surface area rather

than into the active site of an open form of hTK1 homology model, and thus, allowed

effective phosphorylation.

To improve physicochemical properties (e.g. water solubility) of 3CTAs, novel

zwitterionic nido m-carboranyl 3CTAs were synthesized and evaluated as boron-delivery

115 agents for BNCT. All of them were good substrates of hTK1 and showed balanced

hydrophilicity/lipophilicity properties. One zwitterionic nido 3CTA was preferentially

taken up in L929 (wt) tumors implanted in nude mice comparable to N5-2OH and

showed significantly improved water solubility compared with N5-2OH. One

zwitterionic nido m-carboranyl 3CTA was classified as a “2nd Generation 3CTA”, which

has the potential to replace N5-2OH, the current lead compound of our 3CTA library.

The unique properties of the zwitterionic nido m-carborane system were

investigated experimentally and theoretically. Chemical shifts for the nine boron atoms of

the zwitterionic nido m-carborane cluster obtained at the B3LYP/6-31G* and B3LYP/6-

31G** level showed strong correlations with the corresponding experimental values.

Interestingly, it was found that the zwitterionic nido m-carborane system exhibited a

unique reactivity with the carbonyl group of ketones or aldehydes, forming zwitterionic

iminum-substituted carboranes without addition of acid catalyst. The iminium-substituted

carborane exhibited a bathochromic shift in UV experiment due to the conjugation of σ-

aromatic system of anionic nido m-carborane and the double bond of the positively

charged iminium group. The observation was supported by the overlap of alternating-

phase orbitals in HOMO and LUMO and by a small energy gap between HOMO and

LUMO.

In the project related to the development of novel antibiotics, the compound

library composed of known nucleoside-based antiviral and anticancer drugs was screened

to identify a lead compound for the development of potential antibiotics for treatment of anthrax infections. PTAs showed that most of the tested dThd or dUrd analogues of this library were good substrates of BaTK as well as hTK1. However, in vitro toxicity studies

116 of this libray with B. anthracis Sterne showed that most of these compounds are nontoxic except Floxuridine, which inhibits TS in the de novo pathway of DNA biosynthesis, and FLT, which was active only at 20 h incubation.

Twenty dThd-based BaTK/BaTMPK inhibitors were designed and synthesized as potential BaTK and/or BaTMPK inhibitors. The inhibitor structures mimicked dTTP, the endogenous feedback inhibitor of BaTK. The α-phosphate moiety of dTTP was replaced with sulfonamide, amide, urea, thiourea, or triazole group. Some of the urea-, thiourea-, and triazole-substituted dThd inhibitors exhibited relatively low IC50 values at 6 h in

toxicity studies with B. anthracis Sterne. However, most of them were inactive at 20 h

incubation, indicating that they may be converted rapidly into inactive forms under in

vitro toxicity assay conditions.

Overall, the development of novel hTK1-targeting boron-delivery agents for

BNCT was successful and 10B-enriched N5-2OH showed highly promising results in

preclinical neutron irradiation studies. Our design concept for the discovery of novel

antibiotics targeting BaTK and/or BaTMPK was validated and two dTTP-mimicking

inhibitors were selected as lead compounds for further structural optimization.

117 CHAPTER 7

EXPERIMENTAL SECTION

7.1. General Methods

1H-NMR, 13C-NMR, and 11B-NMR spectra were obtained on Bruker 250, 300 or 400

MHz (1H resonance frequency) FT-NMR instruments. Chemical shifts are reported in

parts per million (ppm). The coupling constants are reported in Hertz (Hz). High

resolution electrospray ionization (HR-ESI) mass spectra were recorded on a Micromass

QTOF-Electrospray mass spectrometer and a 3-Tesla Finnigan FTMS-2000 Fourier

Transform mass spectrometer at The Ohio State University Campus Chemical

Instrumentation Center. Electron-impact (EI) mass spectra were obtained with a Kratos

MS-25 mass spectrometer using 70 eV ionization conditions at The Ohio State University

Department of Chemistry. IR spectra were recorded on a Protégé 460 IR Spectrometer

using sodium chloride monocrystal discs. UV spectra were obtained on a Perkin-Elmer

UV/VIS/NIS spectrometer. Optical rotations were measured on a Perkin-Elmer 241

automatic polarimeter. Compound visualization on Silica Gel 60 F254 precoated TLC

plates (0.25 mm layer thickness) (Merck, Darmstadt, Germany) was attained by UV light

and KMnO4 spray. Carborane-containing compounds were selectively visualized by spraying a solution of 0.06% PdCl2 /1% aqueous HCl on TLC plates and subsequent

118 heating to ~ 120 °C. Reagent grade solvents were used for column chromatography using

Silica gel 60, particle size 0.040-0.063 mm (Merck, New Jersey).

Analytical HPLC data of the target compounds were obtained with reversed phase

C8 (RP-8) and C18 (RP-18) 250×4 LiChrosphere 100 Å [5 µm] columns (Merck,) using a Rainin HPLC instrument equipped with a Dynamax DA controller, HPXL pumps, and a

Dynamax UV-1 detector (Rainin Instrument Company Inc., Woburn, MA, USA). HPLC grade water, methanol, and acetonitrile were used as solvents. Four gradient systems and two analytical columns were used as follows.

Method A: Water/methanol gradient (100:0 to 30:70 over 10 min, from 30:70 to

10:90 over 20 min, and from 10:90 to 0:100 over 10 min) with a flow rate of 1 mL/min was applied on RP-18 column.

Method B: Water/acetonitrile gradient (100:0 to 70:30 over 5 min, from 70:30 to

40:60 over 25 min, from 40:60 to 0:100 over 20 min) with a flow rate of 1 mL/min was applied on RP-18 column.

Method C: Water/methanol gradient (100:0 to 70:30 over 10 min, from 70:30 to

90:10 over 20 min, and from 90:10 to 0:100 over 10 min) with a flow rate of 1 mL/min was applied on RP-18 column.

Method D: Water/methanol gradient (100:0 to 70:30 over 10 min, from 70:30 to

90:10 over 20 min, and from 90:10 to 0:100 over 10 min) with a flow rate of 1 mL/min was applied on RP-8 column.

Reagent grade chemicals were obtained from commercial vendors and used as such. Decaborane (99.5% 10B-enriched) and o-carborane (98.0 % 10B-enriched) were purchased from Katchem (Prague, Czech Republic) and osmium tetraoxide from Strem

119 Chemicals (Newburyport, MA). Anhydrous benzene and THF were obtained using

distillation from sodium bezophenone ketyl prior to use. All reactions were carried out

under argon atmosphere. Caution: Decaborane is a highly toxic, impact sensitive

compound, which forms explosive mixtures especially with halogenated materials.

Osmium tetraoxide is a highly toxic and flammable liquid. A careful study of the MSDS is

advisable before usage of both chemicals.

7.2. Chapter 3

(2,3-Propen-1-yl)-o-carborane (3.2.A)

To a solution of o-carborane (4.26 g, 30 mmol) in THF (250 mL) was added a

solution of n-BuLi (12.6 mL, 31.5 mmol, 2.5 M solution in hexanes) at -78 °C. The reaction mixture was gradually warmed to room temperature and stirred for 1 h.

Subsequently, the reaction mixture was cooled down to -78 °C and allyl bromide (2.85 mL, 33 mmol) was added slowly. The mixture was warmed to room temperature and stirred for additional 12 h. Distilled water (50 mL) was added and excess THF was removed under reduced pressure. The residue was extracted with ethylacetate (150 mL ×

2), the combined organic layers were washed with d-HCl solution (100 mL) and brine

(100 mL), and dried over magnesium sulfate. After filtration and evaporation, the residue

was purified by silica gel column chromatography using hexanes as the eluent to give 120 1 compound 3.2.A (3.23 g, 59%). Rf 0.50; H-NMR (CDCl3) δ 2.94 (d, 2H, allyl-CH2, J =

7.5 Hz), 3.56 (br s, 1H, H-Ccarborane), 5.06-5.16 (dq, 1H, C=CH2, J = 16.8, 1.3 Hz), 5.18-

13 5.24 (dq, 1H, C=CH2, J = 10.0, 1.3 Hz) 5.59-5.77 (m, 1H, CH=C); C-NMR (CDCl3) δ

41.65 (CH2), 59.44 (Ccarborane-H), 73.50 (Ccarborane-C), 121.02 (C=C), 131.19 (C=C); MS

+ (HR-ESI) C5H16B10Na (M+Na) calcd 209.2080, found 209.2067.

: 10B-H

(2,3-Propen-1-yl)-o-carborane (3.2.B)

Compound 3.2.B was prepared from 10B-enriched o-carborane in 75% yield

1 adapting the procedure described for compound 3.2.A. Rf 0.50 (only hexanes); H-NMR

(CDCl3) δ 2.93 (d, 2H, allyl-CH2, J = 7.5 Hz), 3.55 (br s, 1H, H-Ccarborane), 5.11 (dd, 1H,

C=CH2, J = 16.8, 1.2 Hz), 5.20 (d, 1H, C=CH2, J = 9.9 Hz), 5.62-5.77 (m, 1H, CH=C);

13 C-NMR (CDCl3) δ 41.66 (CH2), 59.43 (Ccarborane-H), 73.50 (Ccarborane-C), 121.01 (C=C),

+ 131.21 (C=C); MS (HR-EI) C5H16B10 (M ) calcd 176.2569, found 176.2567.

TsO

5-[2-(2,3-Propen-1-yl)-o-carboran-1-yl]pentyl tosylate (3.3.A)

121 To a solution of compound 3.2.A (1.20 g, 6.57 mmol) in benzene (30 mL) was

added a solution of n-BuLi (2.89 mL, 7.23 mmol, 2.5 M in hexanes) at 10 °C over a

period of 20 min. The solution was stirred at room temperature for 1 h and added

dropwise to a solution of 1,5-pentanediol di-p-tosylate (3.25 g, 7.87 mmol) in benzene

(20 mL) at 10 °C. The reaction mixture was stirred for 1 h at the same temperature,

quenched with distilled water (20 mL) and extracted with ethylacetate (40 mL × 3). The

combined organic layers were washed with brine (20 mL) and dried over magnesium

sulfate. After filtration and evaporation, the residue was purified by silica gel column

chromatography using hexanes/ethyl acetate (4:1) as the eluent to give compound 3.3.A

1 (2.08 g, 70%). Rf 0.25; H-NMR (CDCl3) δ 1.29-1.49 (m, 4H, CH2), 1.52-1.69 (m, 2H,

CH2), 2.09-2.12 (m, 2H, CH2-Ccarborane,), 2.43 (s, 3H, CH3), 2.89 (dt, 2H, allyl-CH2, J =

7.2, 1.2 Hz), 3.98 (t, 2H, CH2OTs, J = 6.2 Hz), 5.07 (dq, 1H, C=CH2, J = 16.8, 1.3 Hz),

5.16 (dq, 1H, C=CH2, J = 10.0, 1.3 Hz), 5.64-5.81 (m, 1H, CH=C), 7.33 (d, 2H, ArH, J =

13 13.7 Hz), 7.76 (d, 2H, ArH, J = 13.7 Hz); C-NMR (CDCl3) δ 21.60 (CH2), 25.04 (CH2),

28.33 (CH2), 28.83 (CH2), 34.60 (CH2), 39.28 (CH2), 69.85 (O-CH2), 77.97 (Ccarborane-C),

78.89 (Ccarborane-C), 119.45 (C=C), 127.78 (ArC), 129.86 (ArC), 132.50 (C=C), 132.93

+ (ArC), 144.87 (ArC); MS (HR-ESI) C17H32B10O3S1Na (M+Na) calcd 447.2973, found

447.2983.

TsO

: 10B-H

5-[2-(2,3-Propen-1-yl)-o-carboran-1-yl]pentyl tosylate (3.3.B) 122 Compound 3.3.B was prepared from 3.2.B in 65% yield adapting the procedure

1 described for compound 3.3.A. Rf 0.24 (hexanes/ethylacetate, 4:1); H-NMR (CDCl3) δ

1.23-1.36 (m, 2H, CH2), 1.42-1.50 (m, 2H, CH2), 1.60-1.67 (m, 2H, CH2), 2.07-2.15 (m,

2H, CH2-Ccarborane,), 2.43 (s, 3H, CH3), 2.89 (d, 2H, allyl-CH2, J = 7.1 Hz), 3.99 (t, 2H,

CH2OTs, J = 6.1 Hz), 5.07 (d, 1H, C=CH2, J = 16.9 Hz), 5.16 (d, 1H, C=CH2, J = 10.0

Hz), 5.68-5.77 (m, 1H, CH=C), 7.33 (d, 2H, ArH, J = 8.0 Hz), 7.76 (d, 2H, ArH, J = 8.0

13 Hz); C-NMR (CDCl3) δ 21.66 (CH2), 25.11 (CH2), 28.39 (CH2), 28.88 (CH2), 34.67

(CH2), 39.36 (CH2), 69.84 (O-CH2), 77.97 (Ccarborane-C), 78.85 (Ccarborane-C), 119.47

(C=C), 127.85 (ArC), 129.89 (ArC), 132.58 (C=C), 132.96 (ArC), 144.90 (ArC); MS

+ (HR-ESI) C17H32B10O3S1Na (M+Na) calcd 439.3264, found 439.3271.

O N HO N O O

3-{5-[2-(2,3-Propen-1-yl)-o-carboran-1-yl]pentan-1-yl}thymidine (3.4.A)

To a solution of compound 3.3.A (1.98 g, 4.66 mmol) in DMF/acetone (1:1, 40 mL) were added dThd (2.82 g, 11.64 mmol) and potassium carbonate (2.57 g, 18.59 mmol). The mixture solution was stirred for 96 h at 35 °C, filtered, and the filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography using dichloromethane/acetone (4:1) as the eluent to give compound 3.4.A (1.21 g, 52 %).

1 Rf 0.21; H-NMR (CD3OD) δ 1.31-1.40 (m, 2H, CH2), 1.52-1.67 (m, 4H, CH2), 1.89 (d,

123 3H, CH3, J = 1.2 Hz), 2.11-2.31 (m, 4H, H-2´ and CH2-Ccarborane), 3.05 (dt, 2H, allyl-CH2,

J = 7.2 Hz, 1.2 Hz), 3.71 (dd, 1H, H-5´, J = 12.0, 3.1 Hz), 3.79 (dd, 1H, H-5´, J = 12.00,

3.7 Hz), 3.87-3.93 (m, 3H, H-4´ and CH2N), 4.36-4.41 (m, 1H, H-3´), 5.12-5.16 (m, 1H,

C=CH2), 5.19-5.20 (m, 1H, C=CH2) 5.72-5.86 (m, 1H, CH=C), 6.29 (t, 1H, H-1´, J = 6.9

13 Hz), 7.82 (d, 1H, H-6, J = 1.2 Hz); C-NMR (CD3OD) δ 13.23 (CH3), 27.21 (CH2),

27.88 (CH2), 30.32 (CH2), 35.62 (CH2), 40.08 (CH2), 41.34 (CH2), 41.85 (CH2), 62.75

(C-5´), 72.09 (C-3´), 80.03 (Ccarborane-C), 81.28 (Ccarborane-C), 87.07 (C-1´), 88.85 (C-4´),

110.68 (C-5) 119.92 (C=C), 134.31(C=C), 136.43 (C-6), 152.28 (C-2), 165.34 (C-4); MS

+ (HR-ESI) C20H38B10N2O5Na (M+Na) calcd 517.3676, found 517.3651.

O N HO N O O

OH : 10B-H

3-{5-[2-(2,3-Propen-1-yl)-o-carboran-1-yl]pentan-1-yl}thymidine (3.4.B)

Compound 3.4.B was prepared from 3.3.B in 79% yield adapting the procedure

1 described for compound 3.4.A. Rf 0.21 (dichloromethane/acetone, 4:1); H-NMR

(CD3OD) δ 1.31-1.38 (m, 2H, CH2), 1.54-1.65 (m, 4H, CH2), 1.89 (d, 3H, CH3, J = 1.1

Hz), 2.12-2.30 (m, 4H, H-2´ and CH2-Ccarborane), 3.05 (d, 2H, allyl-CH2, J = 7.3 Hz), 3.71

(dd, 1H, H-5´, J = 12.0, 3.1 Hz), 3.79 (dd, 1H, H-5´, J = 12.00, 3.6 Hz), 3.88-3.92 (m, 3H,

H-4´ and CH2N), 4.37-4.40 (m, 1H, H-3´), 5.14-5.19 (m, 2H, C=CH2) 5.76-5.86 (m, 1H,

13 CH=C), 6.29 (t, 1H, H-1´, J = 6.9 Hz), 7.83 (d, 1H, H-6, J = 1.1 Hz); C NMR (CD3OD)

124 δ 13.22 (CH3), 27.21 (CH2), 27.88 (CH2), 30.33 (CH2), 35.63 (CH2), 40.09 (CH2), 41.34

(CH2), 41.84 (CH2), 62.76 (C-5´), 72.11 (C-3´), 80.08 (Ccarborane-C), 81.32 (Ccarborane-C),

87.08 (C-1´), 88.87 (C-4´), 110.70 (C-5) 119.90 (C=C), 134.35 (C=C), 136.45 (C-6),

+ 152.31 (C-2), 165.38 (C-4); MS (HR-ESI) C20H38B10N2O5Na (M+Na) calcd 509.3972,

found 509.3969.

O N OH HO N O OH O

3-{5-[2-(2,3-Dihydroxyprop-1-yl)-o-carboran-1-yl]pentan-1-yl}thymidine (3.5.A)

To a solution of compound 3.4.A (980 mg, 1.98 mmol) and NMO (500 mg, 4.26

mmol) in 1,4-dioxane (20 mL) was added an aqueous solution of OsO4 (0.1 g in 10 mL

H2O). The reaction mixture was protected from light by covering the reaction flask with

aluminum foil, stirred at room temperature for 6 h, quenched with a concentrated aqueous

solution of Na2S2O3 (500 mg, 3.16 mmol), and excess solvents were removed under

reduced pressure. The residue was purified by silica gel column chromatography using

dichloromethane/acetone (1:1) as the eluent to give compound 3.5.A (400 mg, 40 %). Rf

25 1 0.30; [α]D -5.1 (c = 0.10; MeOH); H-NMR (CD3OD) δ 1.28-1.39 (m, 2H, CH2), 1.52-

1.67 (m, 4H, CH2), 1.89 (d, 3H, CH3, J = 1.2 Hz), 2.12-2.36 (m, 5H, H-2´ and CH2-

Ccarborane, and CH(OH)-CH2-Ccarborane), 2.56 (dd, 1H, CH(OH)-CH2-Ccarborane, J = 15.8, 1.7

Hz), 3.33 (dd, 1H, CH2OH, J = 11.0, 6.5 Hz) 3.47 (dd, 1H, CH2OH, J = 11.0, 5.3 Hz),

3.71 (dd, 1H, H-5´, J = 12.0, 3.7 Hz), 3.74-3.80 (m, 1H, CH(OH)-CH2OH), 3.80 (dd, 1H, 125 H-5´, J = 12.0, 3.1 Hz), 3.87-3.93 (m, 3H, H-4´ and CH2N), 4.36-4.41 (m, 1H, H-3´),

13 6.29 (t, 1H, H-1´, J = 6.9 Hz), 7.83 (d, 1H, H-6, J = 1.2 Hz); C-NMR (CD3OD) δ 13.23

(CH3), 27.29 (CH2), 27.90 (CH2), 30.38 (CH2), 35.80 (C-Ccarborane), 39.89 (C-Ccarborane),

41.34 (C-2´), 41.92 (CH2N), 62.76 (C-5´), 66.91 (O-CH2), 72.11 (O-CH), 72.21 (C-3´),

80.44 (Ccarborane-C), 81.89 (Ccarborane-C), 87.10 (C-1´), 88.87 (C-4´), 110.71 (C-5), 136.47

11 (C-6), 152.33 (C-2), 165.43 (C-4); B-NMR (CD3OD) δ -7.4 (8B), -1.9 (2B); IR 1054,

1099, 1269, 1472, 1627, 1666, 1694, 2580 (B-H), 2935, 3405; MS (HR-ESI)

+ C20H40B10N2O7Na (M+Na) calcd 551.3747, found 551.3728; HPLC retention time: 26.9 min (Method A), 21.1 min (Method B).

O N OH HO N O OH O : 10B-H OH

3-{5-[2-(2,3-Dihydroxyprop-1-yl)-o-carboran-1-yl]pentan-1-yl}thymidine (3.5.B)

Compound 3.5.B was prepared from 3.4.B in 40% yield adapting the procedure

1 described for compound 3.5.A. Rf 0.30 (dichloromethane/acetone, 1:1); H-NMR

(CD3OD) δ 1.28-1.39 (m, 2H, CH2), 1.52-1.67 (m, 4H, CH2), 1.89 (d, 3H, CH3, J = 1.1

Hz), 2.17-2.39 (m, 5H, H-2´ and CH2-Ccarborane, and CH(OH)-CH2-Ccarborane), 2.56 (dd, 1H,

CH(OH)-CH2-Ccarborane, J = 15.8, 1.6 Hz), 3.33 (dd, 1H, CH2OH, J = 11.0, 6.5 Hz), 3.46

(dd, 1H, CH2OH, J = 11.0, 5.3 Hz), 3.72 (dd, 1H, H-5´, J = 12.0, 3.7 Hz), 3.76-3.80 (m,

1H, CH(OH)-CH2OH), 3.79 (dd, 1H, H-5´, J = 12.0, 3.1 Hz), 3.81-3.92 (m, 3H, H-4´ and

126 CH2N), 4.37-4.40 (m, 1H, H-3´), 6.30 (t, 1H, H-1´, J = 6.7 Hz), 7.83 (d, 1H, H-6, J = 1.1

13 Hz); C-NMR (CD3OD) δ 13.21 (CH3), 27.29 (CH2), 27.91 (CH2), 30.39 (CH2), 35.83

(CH2), 39.92 (CH2), 41.34 (CH2), 41.92 (CH2), 62.77 (C-5´), 66.92 (O-CH2), 72.13 (O-

CH2), 72.23 (C-3´), 80.49 (Ccarborane-C), 81.94 (Ccarborane-C), 87.11 (C-1´), 88.89 (C-4´),

110.72 (C-5), 136.49 (C-6), 152.34 (C-2), 165.45 (C-4); IR 1054, 1097, 1267, 1470, 1625,

+ 1664, 1690, 2587 (B-H), 2926, 3385; MS (HR-ESI) C20H40B10N2O7Na (M+Na) calcd

543.4027, found 543.4003; HPLC retention time: 26.5 min (Method A), 21.0 min

(Method B).

Si H

10 : B-H

tert-Butyldimethylsilyl-o-carborane (3.6)

Method I: Decaborane (635 mg, 5.21 mmol) was dissolved in acetonitrile (10 mL)

and refluxed at 80 °C for 30 min. The solution turned yellow due to the formation of the

decaborane-acetonitrile adducts. To this solution was added tert-butylsilylacetylene (0.75

mL, 3.85 mmol) in toluene (10 mL) and the resulting reaction mixture was refluxed for

24 h. The dark brown solution was cooled to room temperature and methanol (1 mL) was

added. After evaporation, the residue was purified by silica gel column chromatography

using hexanes as the eluent to give the compound 3.6 (299 mg, 23%).

Method II: To a solution of 1-butyl-3-methylimidiazolium chloride (990 mg) in

toluene (10 mL) was added decaborane (122 mg, 1 mmol) and tert-butylsilylacetylene

127 (0.56 mL, 3 mmol) at 120 °C under argon atmosphere. The mixture was stirred

vigorously at the same temperature for 10 min. After removal of toluene, the residue was

purified by silica gel column chromatography using hexane as the eluent to give the

1 compound 3.6 (125 mg, 50%). Rf 0.50 (100% pentane); H-NMR (CDCl3) δ 0.91 (s, 6H,

13 -Si(CH3)2), 1.22 (s, 9H, C(CH3)3), 3.56 (br s, 1H, H-Ccarborane); C-NMR(CDCl3) δ -5.34

(CH3), 13.21 (CH3), 28.87 (CH2), 59.44 (Ccarborane-H), 73.50 (Ccarborane-C); MS(HR-EI)

+ C8H26B10Si (M ) calcd 250.8765, found 250.8654.

10 : B-H

1-(tert-Butyldimethylsilyl)-2-(2,3-propen-1-yl)-o-carborane (3.7)

To a solution of 3.6 (290 mg, 1.16 mmol) in THF (50 mL) was added n-BuLi

(0.464 mL, 1.16 mmol, 2.5 M solution in hexanes) at -78 °C over a period of 10 min. The solution was gradually warmed to room temperature and was stirred for 1 h. Allyl

bromide (0.10 mL, 1.2 mmol) was added to the solution at -78 °C and the reaction

mixture was stirred at room temperature for 12 h. Distilled water (50 mL) was added and

THF was removed under reduced pressure. The residue was extracted with ethylacetate

(30 mL × 4). The combined organic layers were washed with d-HCl solution (100 mL)

and brine (100 mL), and dried over MgSO4. After filtration and evaporation, the residue

was purified by silica gel column chromatography using hexanes as the eluent to give

1 compound 3.7 (201 mg, 60 %). Rf 0.60 (100% hexanes); H-NMR (CDCl3) δ 0.92 (s, 6H,

128 -Si(CH3)2), 1.23 (s, 9H, C(CH3)3), 2.93 (d, 2H, allyl-CH2, J = 7.5 Hz), 5.06-5.16 (m, 1H,

13 C=CH2), 5.18-5.24 (m, 1H, C=CH2), 5.59-5.77 (m, 1H, CH=C); C-NMR (CDCl3) δ -

5.28 (CH3), 13.21 (CH3), 21.90 (CH3), 28.87 (CH2), 41.65 (CH2), 72.76 (Ccarborane-C),

+ 73.50 (Ccarborane-C), 121.02 (C=C), 131.19 (C=C); MS (HR-EI) C11H30B10Si (M ) calcd

290.2080, found 290.2067.

: 10B-H

(2,3-Propen-1-yl)-o-carborane (3.2.B)

To a solution of compound 3.7 (170 mg, 0.96 mmol) in THF (10 mL) was added

TBAF (1.5 mL, 1.0 M in THF) at -78 °C. The mixture was stirred at room temperature

for 2 h, and then poured into ice-water (30 mL), extracted with diethyl ether (30 mL × 3).

The combined organic layers were washed with brine and dried over MgSO4. After filtration and evaporation, crude compound 3.2.B (135 mg, 80%) was obtained.

Analytical data were identical to those for the 3.2.B preparation described above.

H O O

(R)-4-(o-Carboran-1-yl)methyl-2,2-dimethyl-1,3-dioxolane (3.8.A)

To a solution of o-carborane (213 mg, 1.5 mmol) in benzene (5 mL) was added a

solution of n-BuLi (0.66 mL, 1.65 mmol, 2.5 M solution in hexanes) at 0 °C. The 129 solution was first stirred for 30 min at 0 °C and subsequently for 30 min at room

temperature. The reaction mixture was again cooled to 5 °C and (S)-(+)-2,2-dimethyl-1,3-

dioxolane-4-ylmethyl p-tosylate (429 mg, 1.5 mmol) in benzene (10 mL) was added

dropwise. The reaction mixture was stirred at room temperature for 14 h. Subsequently,

distilled water (10 mL) was added and excess benzene was removed under reduced

pressure. The residue was extracted with ethylacetate (50 mL), the organic layer was

washed with d-HCl solution (20 mL) and brine (20 mL) and dried over MgSO4. After filtration and evaporation, the residue was purified by silica gel columns chromatography using hexanes/ ethylacetate (25:1) as the eluent to give compound 3.8.A (242 mg, 63%).

1 Rf 0.12; H-NMR (CDCl3) δ 1.31 (s, 3H, CH3), 1.37 (s, 3H, CH3), 2.39 (dd, 1H, CH2, J =

15.1, 3.4 Hz), 2.47 (dd, 1H, CH2, J = 15.1, 9.1 Hz), 3.47 (dd, 1H, CH2 J = 8.4, 5.9 Hz),

4.02 (br s, 1H, H-Ccarborane), 4.05 (dd, 1H, CH2, J = 8.4, 6.2 Hz), 4.17-4.23 (m, 1H, CH);

13 C-NMR (CDCl3) δ 25.4 (CH3), 26.9 (CH3), 42.0 (CH2-Ccarborane), 59.8 (H-Ccarborane),

68.7 (CH2), 72.4 (CH2-Ccarborane), 73.8 (CH), 110.2 [C(CH3)2]

H O O

(S)-4-(o-Carboran-1-yl)methyl-2,2-dimethyl-1,3-dioxolane (3.8.B)

Compound 3.8.B was prepared from (R)-(-)-2,2-dimethyl-1,3-dioxolane-4- ylmethyl p-tosylate in 60% yield adapting the procedure described for compound 3.8.A.

1 Rf 0.12 (hexanes/ethylacetate, 25:1); H-NMR (CDCl3) δ 1.31 (s, 3H, CH3), 1.37 (s, 3H,

CH3), 2.39 (dd, 1H, CH2, J = 15.1, 3.5 Hz), 2.47 (dd, 1H, CH2, J = 15.1, 9.1 Hz), 3.47 (dd, 130 1H, CH2 J = 8.4, 5.9 Hz), 4.02 (br s, 1H, H-Ccarborane), 4.05 (dd, 1H, CH2, J = 8.4, 6.2 Hz),

13 4.18-4.23 (m, 1H, CH); C-NMR (CDCl3) δ 25.4 (CH3), 26.9 (CH3), 42.0 (CH2-Ccarborane),

59.8 (H-Ccarborane), 68.7 (CH2), 72.4 (CH2-Ccarborane), 73.8 (CH), 110.2 [C(CH3)2]

H O O

4-(o-Carboran-1-yl)methyl-2,2-dimethyl-1,3-dioxolane (3.8.C)

Compound 3.8.C was prepared from 2,2-dimethyl-1,3-dioxolane-4-ylmethyl p- tosylate in 65% yield adapting the procedure described for compound 3.8.A. Rf 0.12

1 (hexanes/ ethylacetate, 25:1); H-NMR (CDCl3) δ 1.31 (s, 3H, CH3), 1.36 (s, 3H, CH3),

2.38 (dd, 1H, CH2, J = 15.2, 3.9 Hz), 2.47 (dd, 1H, CH2, J = 15.2, 8.8 Hz), 3.47 (dd, 1H,

CH2, J = 8.4, 5.9 Hz), 4.02 (br s, 1H, H-Ccarborane), 4.05 (dd, 1H, CH2, J = 8.4, 6.1 Hz),

13 4.18-4.23 (m, 1H, CH); C-NMR (CDCl3) δ 25.8 (CH3), 27.3 (CH3), 42.4 (CH2-Ccarborane),

60.3 (H-Ccarborane), 69.1 (CH2), 72.8 (CH2-Ccarborane), 74.2 (CH), 110.6 [C(CH3)2]

O O

4-(m-Carboran-1-yl)methyl-2,2-dimethyl-1,3-dioxolane (3.8.D)

Compound 3.8.D was prepared from m-carborane and 2,2-dimethyl-1,3-

dioxolane-4-ylmethyl p-tosylate in 59% yield adapting the procedure described for

1 compound 3.8.A. Rf 0.16 (hexanes/ ethylacetate, 15:1); H-NMR (CDCl3) δ 1.30 (s, 3H, 131 CH3), 1.34 (s, 3H, CH3), 2.07 (dd, 1H, CH2-Ccarborane, J = 14.7, 5.3 Hz), 2.29 (dd, 1H,

CH2-Ccarborane, J = 14.7, 5.6 Hz), 2.92 (br s, 1H, H-Ccarborane), 3.42-3.48 (m, 1H, CH2),

13 4.01-4.07 (m, 2H); C-NMR (CDCl3) δ 25.35 (CH3), 26.86 (CH3), 41.18 (CH2-Ccarborane),

55.26 (H-Ccarborane), 69.00 (CH2), 72.59 (CH2-Ccarborane), 74.66 (CH), 109.13 [C(CH3)2]

4-(p-Carboran-1-yl)methyl-2,2-dimethyl-1,3-dioxolane (3.8.E)

Compound 3.8.E was prepared from p-carborane and 2,2-dimethyl-1,3-dioxolane-

4-ylmethyl p-tosylate in 50% yield adapting the procedure described for compound 3.8.A.

1 Rf 0.18 (hexanes/ ethylacetate, 15:1); H-NMR (CDCl3) δ 1.26 (s, 3H, CH3), 1.29 (s, 3H,

CH3), 1.76 (dd, 1H, CH2-Ccarborane, J = 14.9, 6.8 Hz), 1.98 (dd, 1H, CH2-Ccarborane, J =

14.9, 5.4 Hz), 2.63 (br s, 1H, H-Ccarborane), 3.34 (dd, 1H, CH2, J = 8.1, 5.9 Hz), 3.81-3.86

13 (m, 1H, CH), 3.95 (dd, 1H, CH2, J = 8.1, 5.9 Hz); C-NMR (CDCl3) δ 25.71 (CH3),

27.21 (CH3), 43.42 (CH2-Ccarborane), 59.24 (H-Ccarborane), 69.42 (CH2), 74.81 (CH), 81.33

(CH2-Ccarborane), 109.18 [C(CH3)2]

TsO O O

132 (R)-5-[2-(2,3-Isopropylidenedioxyprop-1-yl)-o-carboran-1-yl]pentyl tosylate (3.9.A)

To a solution of compound 3.8.A (129 mg, 0.5 mmol) in benzene (5 mL) was added n-BuLi (0.22 mL, 0.55 mmol, 2.5 M solution in hexanes) at 5 °C over a period of

20 min. The solution was first stirred at the same temperature for 30 min and then at room temperature for 30 min. The solution was then slowly added to a solution of 1,5- pentanediol di-p-tosylate (330 mg, 0.80 mmol) in benzene (10 mL) at 5 °C (ice bath).

The reaction mixture was stirred for 1 h at the same temperature. Distilled water (5 mL)

was added and the reaction mixture was extracted with ethylacetate (15 mL × 3). The

combined organic layers were washed with brine (20 mL) and dried over magnesium

sulfate. After filtration and evaporation, the residue was purified by silica gel column

chromatography using hexanes/ethylacetate (4:1) as the eluent to give compound 3.9.A

1 (144 mg, 58 %). Rf 0.21; H-NMR (CDCl3) δ 1.31 (s, 3H, CH3), 1.35 (s, 3H, CH3), 1.41-

1.51 (m, 2H, CH2), 1.60-1.67 (m, 2H, CH2), 2.06-2.23 (m, 4H, CH2 and CH2-Ccarborane),

2.34 (dd, 1H, CH2-Ccarborane, J = 15.5, 5.1 Hz), 2.41 (dd, J = 15.5, 6.5 Hz), 2.43 (s, 3H,

CH3), 3.54 (dd, 1H, CH2, J = 8.3, 6.5 Hz), 3.99 (t, 2H, OCH2, J = 6.2 Hz), 4.11 (dd, 1H,

CH2, J = 8.3, 6.0 Hz), 4.19-4.25 (m, 1H, CH), 7.32 (d, 2H, ArH, J = 8.1 Hz), 7.75 (d, 2H,

13 ArH, J = 8.1 Hz); C-NMR (CDCl3) δ 21.62 (CH3), 25.05 (CH3), 25.25 (CH3), 26.79

(CH2), 28.35 (CH2), 28.87 (CH2), 34.81 (CH2-Ccarborane), 39.40 (CH2-Ccarborane), 69.04

(CH2), 69.81 (CH2OTs), 74.39 (CH), 76.86 (C-Ccarborane), 79.62 (C-Ccarborane), 109.54

[C(CH3)2), 127.80 (ArC), 129.87 (ArC), 132.90 (ArC), 144.86 (ArC); MS (HR-ESI)

+ C20H38B10O5S1Na (M+Na) calcd 521.3352, found 521.3347.

133 TsO O O

(S)-5-[2-(2,3-Isopropylidenedioxyprop-1-yl)-o-carboran-1-yl]pentyl tosylate (3.9.B)

Compound 3.9.B was prepared from 3.8.B in 62% yield adapting the procedure

1 described for compound 3.9.A. Rf 0.21 (hexanes/ethylacetate, 4:1); H-NMR (CDCl3) δ

1.32 (s, 3H, CH3), 1.36 (s, 3H, CH3), 1.42-1.51 (m, 2H, CH2), 1.61-1.68 (m, 2H, CH2),

2.02-2.23 (m, 4H, CH2 and CH2-Ccarborane), 2.34 (dd, 1H, CH2-Ccarborane, J = 15.4, 5.2 Hz),

2.42 (dd, 1H, CH2-Ccarborane, J = 15.4, 6.5 Hz), 2.45 (s, 3H, CH3), 3.54 (dd, 1H, CH2, J =

8.3, 6.4 Hz), 3.99 (t, 2H, OCH2, J = 6.2 Hz), 4.12 (dd, 1H, CH2, J = 8.3, 6.0 Hz), 4.19-

4.25 (m, 1H, CH), 7.32 (d, 2H, ArH, J = 8.1 Hz), 7.75 (d, 2H, ArH, J = 8.1 Hz); 13C-

NMR (CDCl3) δ 21.64 (CH3), 25.07 (CH3), 25.26 (CH3), 26.81 (CH2), 28.37 (CH2),

28.89 (CH2), 34.83 (CH2-Ccarborane), 39.42 (CH2-Ccarborane), 69.07 (CH2), 69.81 (CH2OTs),

74.41 (CH), 76.84 (C-Ccarborane), 79.62 (C-Ccarborane), 109.55 [C(CH3)2], 127.82 (ArC),

+ 129.87 (ArC), 132.92 (ArC), 144.87 (ArC); MS (HR-ESI) C20H38B10O5S1Na (M+Na) calcd 521.3352, found 521.3345.

TsO O O

5-[2-(2,3-Isopropylidenedioxyprop-1-yl)-o-carboran-1-yl]pentyl tosylate (3.9.C)

Compound 3.9.C was prepared from 3.8.C in 60% yield adapting the procedure

1 described for compound 3.9.A. Rf 0.21 (hexanes/ethylacetate, 4:1); H-NMR (CDCl3) δ 134 1.32 (s, 3H, CH3), 1.36 (s, 3H, CH3), 1.42-1.51 (m, 2H, CH2), 1.61-1.68 (m, 2H, CH2),

2.02-2.23 (m, 4H, CH2 and CH2-Ccarborane), 2.34 (dd, 1H, CH2-Ccarborane, J = 15.4, 5.2 Hz),

2.42 (dd, 1H, CH2-Ccarborane, J = 15.4, 6.4 Hz), 2.44 (s, 3H, CH3), 3.55 (dd, 1H, CH2, J =

8.2, 6.5 Hz), 3.99 (t, 2H, OCH2, J = 6.1 Hz), 4.12 (dd, 1H, CH2, J = 8.2, 6.1 Hz), 4.20-

4.26 (m, 1H, CH), 7.33 (d, 2H, ArH, J = 8.1 Hz), 7.76 (d, 2H, ArH, J = 8.1 Hz); 13C-

NMR (CDCl3) δ 21.7 (CH3), 25.0 (CH3), 25.3 (CH3), 26.8 (CH2), 28.7 (CH2), 30.9 (CH2),

34.8 (CH2-Ccarborane), 39.5 (CH2-Ccarborane), 69.1 (CH2), 70.8 (CH2OTs), 74.5 (CH), 109.6

[C(CH3)2], 127.8 (ArC), 129.9 (ArC), 133.0 (ArC), 144.9 (ArC); MS (HR-ESI)

+ C20H38B10O5S1Na (M+Na) calcd 521.3352, found 521.3365.

TsO

O O

5-[2-(2,3-Isopropylidenedioxyprop-1-yl)-m-carboran-1-yl]pentyl tosylate (3.9.D)

Compound 3.9.D was prepared from 3.8.D in 64% yield adapting the procedure

1 described for compound 3.9.A. Rf 0.21 (hexanes/ethylacetate, 4:1); H-NMR (CDCl3) δ

1.20-1.28 (m, 4H, CH2), 1.30 (s, 3H, CH3), 1.34 (s, 3H, CH3), 1.55-1.60 (m, 2H, CH2),

1.81-1.85 (m, 2H, CH2-Ccarborane), 2.04 (dd, 1H, CH2-Ccarborane, J = 14.7, 5.4 Hz), 2.27 (dd,

1H, CH2-Ccarborane, J = 14.7, 5.6 Hz), 2.44 (s, 3H, CH3), 3.42-3.48 (m, 1H, CH2), 3.97 (t,

2H, OCH2, J = 6.3 Hz), 4.00-4.05 (m, 2H), 7.32 (d, 2H, ArH, J = 8.2 Hz), 7.75 (d, 2H,

13 ArH, J = 8.2 Hz); C-NMR (CDCl3) δ 21.64 (CH3), 24.67 (CH2), 25.36 (CH3), 26.88

(CH3), 28.38 (CH2), 29.16 (CH2), 36.62 (CH2-Ccarborane), 41.18 (CH2-Ccarborane), 69.02

135 (CH2), 70.2 (CH2OTs), 72.06 (C-Ccarborane), 74.65 (CH), 76.06 (C-Ccarborane), 109.08

(C(CH3)2), 127.84 (Ar-C), 129.84 (Ar-C), 133.01 (Ar-C), 144.78 (Ar-C); MS (HR-ESI)

+ C20H38B10O5S1Na (M+Na) calcd 521.3352, found 521.3336.

TsO

5-[2-(2,3-Isopropylidenedioxyprop-1-yl)-p-carboran-1-yl]pentyl tosylate (3.9.E)

Compound 3.9.E was prepared from 3.8.E in 65% yield adapting the procedure

1 described for compound 3.9.A. Rf 0.21 (hexanes/ethylacetate, 4:1); H-NMR (CDCl3) δ

0.99-1.13 (m, 4H, CH2), 1.26 (s, 3H, CH3), 1.29 (s, 3H, CH3), 1.48-1.55 (m, 4H, CH2 and

CH2-Ccarborane), 1.75 (dd, 1H, CH2-Ccarborane, J = 14.9, 7.1 Hz), 1.98 (dd, 1H, CH2-Ccarborane,

J = 14.9, 5.4 Hz), 2.43 (s, 3H, CH3), 3.34 (dd, 1H, CH2-Ccarborane, J = 8.2, 6.1 Hz), 3.78-

3.86 (m, 1H, CH), 3.92 (t, 2H, OCH2, J = 6.4 Hz), 3.99-4.03 (m, 1H, CH2), 7.32 (d, 2H,

13 ArH, J = 8.2 Hz), 7.75 (d, 2H, ArH, J = 8.2 Hz); C-NMR (CDCl3) δ 21.63 (CH3),

24.82 (CH2), 25.30 (CH3), 26.81 (CH3), 28.66 (CH2), 30.90 (CH2), 37.45 (CH2- Ccarborane),

41.32 (CH2-Ccarborane), 69.04 (CH2), 70.03 (CH2OTs), 74.55 (CH), 75.12 (C-Ccarborane),

78.15 (C-Ccarborane), 108.78 (C(CH3)2), 127.84 (Ar-C), 129.83 (Ar-C), 133.13 (Ar-C),

+ 144.73 (Ar-C); MS (HR-ESI) C20H38B10O5S1Na (M+Na) calcd 521.3352, found

521.3339.

136 O

N O HO N O O O

(R)-3-{5-[2-(2,3-Isopropylidenedioxyprop-1-yl)-o-carboran-1-yl]pentan-1-

yl}thymidine (3.10.A)

To a solution of compound 3.9.A (125 mg, 0.25 mmol) in a mixture of DMF and

acetone (10 mL, 1:1) was added dThd (150 mg, 0.62 mmol) and potassium carbonate

(150 mg, 1.13 mmol). The reaction mixture was stirred at 50 °C for 48 h. The reaction

mixture was filtered and the filtrate was concentrated under reduced pressure. The

residue was purified by silica gel columns chromatography using ethylacetate/methanol

1 (20:1) as the eluent to give compound 3.10.A (88 mg, 62%). Rf 0.29; H-NMR (MeOH-

4 d ) δ 1.30-1.35 (m, 2H, CH2) 1.32 (s, 3H, CH3), 1.34 (s, 3H, CH3), 1.54-1.65 (m, 4H,

CH2), 1.89 (d, 3H, CH3, J = 1.1 Hz), 2.15-2.28 (m, 2H, H-2´), 2.28-2.34 (m, 2H, CH2-

Ccarborane), 2.51 (dd, 1H, CH2-Ccarborane, J = 15.9, 7.9 Hz), 2.58 (dd, 1H, CH2-Ccarborane, J =

15.9, 3.5 Hz), 3.53 (dd, 1H, CH2, J = 8.2, 7.2 Hz), 3.72 (dd, 1H, H-5´, J = 12.0, 3.7 Hz),

3.79 (dd, 1H, H-5´, J = 12.0, 3.7 Hz), 3.88-3.92 (m, 3H, H-4´ and CH2N), 4.10 (dd, 1H,

CH2, J = 8.3, 6.1 Hz), 4.23-4.29 (m, 1H, CH), 4.37-4.40 (m, 1H, H-3´), 6.30 (t, 1H, H-1´,

13 4 J = 6.8 Hz), 7.83 (d, 1H, H-6, J = 1.1 Hz); C-NMR (MeOH-d ) δ 13.24 (CH3), 25.85

(CH3), 27.20 (CH3), 27.26 (CH2), 27.92 (CH2), 30.34 (CH2), 35.73 (CH2-Ccarborane), 40.25

(CH2-Ccarborane ), 41.35 (C-2´), 41.87 (CH2-N), 62.76 (C-5´), 69.99 (CH2), 72.11 (C-3´),

75.95 (CH), 79.53 (CH2-Ccarborane), 81.77 (CH2-Ccarborane), 87.05 (C-1´), 88.86 (C-4´),

137 110.70 (C-5), 110.89 [C(CH3)2], 136.44 (C-6), 152.30 (C-2), 165.36 (C-4); MS (HR-ESI)

+ C23H44B10N2O7Na (M+Na) calcd 591.4061, found 591.4063.

N O HO N O O O

(S)-3-{5-[2-(2,3-Isopropylidenedioxyprop-1-yl)-o-carboran-1-yl]pentan-1-

yl}thymidine (3.10.B)

Compound 3.10.B was prepared from 3.9.B in 65% yield adapting the procedure

1 described for compound 3.10.A. Rf 0.29 (ethylacetate/methanol, 20:1); H-NMR (MeOH-

4 d ) δ 1.30-1.35 (m, 2H, CH2) 1.32 (s, 3H, CH3), 1.34 (s, 3H, CH3), 1.54-1.65 (m, 4H,

CH2), 1.89 (d, 3H, CH3, J = 1.1 Hz), 2.14-2.28 (m, 2H, H-2´), 2.28-2.34 (m, 2H, CH2-

Ccarborane), 2.51 (dd, 1H, CH2-Ccarborane, J = 15.9, 7.9 Hz), 2.58 (dd, 1H, CH2-Ccarborane, J =

15.9, 3.5 Hz), 3.53 (dd, 1H, CH2, J = 8.2, 7.2 Hz), 3.71 (dd, 1H, H-5´, J = 12.0, 3.7 Hz),

3.79 (dd, 1H, H-5´, J = 12.0, 3.7 Hz), 3.88-3.92 (m, 3H, H-4´ and CH2N), 4.10 (dd, 1H,

CH2, J = 8.3, 5.9 Hz), 4.23-4.29 (m, 1H, CH), 4.37-4.40 (m, 1H, H-3´), 6.30 (t, 1H, H-1´,

13 4 J = 6.8 Hz), 7.83 (d, 1H, H-6, J = 1.1 Hz); C-NMR (MeOH-d ) δ 13.23 (CH3), 25.85

(CH3), 27.20 (CH3), 27.26 (CH2), 27.93 (CH2), 30.35 (CH2), 35.74 (CH2-Ccarborane), 40.25

(CH2-Ccarborane), 41.35 (C-2´), 41.87 (CH2-N), 62.76 (C-5´), 69.99 (CH2), 72.11 (C-3´),

75.96 (CH), 79.54 (CH2-Ccarborane), 81.78 (CH2-Ccarborane), 87.06 (C-1´), 88.87 (C-4´),

138 110.70 (C-5), 110.90 [C(CH3)2], 136.45 (C-6), 152.31 (C-2), 165.37 (C-4); MS (HR-ESI)

+ C23H44B10N2O7Na (M+Na) calcd 591.4061, found 591.4067.

N O HO N O O O

3-{5-[2-(2,3-Isopropylidenedioxyprop-1-yl)-o-carboran-1-yl]pentan-1-yl}thymidine

(3.10.C)

Compound 3.10.C was prepared from 3.9.C in 60% yield adapting the procedure

1 described for compound 3.10.A. Rf 0.29 (ethylacetate/methanol, 20:1); H-NMR (MeOH-

4 d ) δ 1.30-1.35 (m, 2H, CH2) 1.32 (s, 3H, CH3), 1.34 (s, 3H, CH3), 1.54-1.65 (m, 4H,

CH2), 1.89 (d, 3H, CH3, J = 1.2 Hz), 2.14-2.28 (m, 2H, H-2´), 2.28-2.34 (m, 2H, CH2-

Ccarborane), 2.51 (dd, 1H, CH2-Ccarborane, J = 15.9, 7.9 Hz), 2.58 (dd, 1H, CH2-Ccarborane, J =

15.9, 3.4 Hz), 3.54 (dd, 1H, CH2, J = 8.2, 6.5 Hz), 3.71 (dd, 1H, H-5´, J = 12.0, 3.7 Hz),

3.79 (dd, 1H, H-5´, J = 12.0,3.7 Hz), 3.88-3.91 (m, 3H, H-4´ and CH2N), 4.09 (dd, 1H,

CH2, J = 8.1, 6.1 Hz), 4.23-4.29 (m, 1H, CH), 4.37-4.40 (m, 1H, H-3´), 6.30 (t, 1H, H-1´,

13 4 J = 6.8 Hz), 7.83 (d, 1H, H-6, J = 1.2 Hz); C-NMR (MeOH-d ) δ 13.22 (CH3), 25.84

(CH3), 27.19 (CH3), 27.26 (CH2), 27.92 (CH2), 30.35 (CH2), 35.74 (CH2-Ccarborane), 40.25

(CH2-Ccarborane ), 41.35 (C-2´), 41.87 (CH2-N), 62.76 (C-5´), 70.00 (CH2), 72.12 (C-3´),

75.97 (CH), 79.54 (CH2-Ccarborane), 81.78 (CH2-Ccarborane), 87.06 (C-1´), 88.88 (C-4´),

139 110.70 (C-5), 110.90 (C(CH3)2), 136.49 (C-6), 152.32 (C-2), 165.38 (C-4); MS (HR-ESI)

+ C23H44B10N2O7Na (M+Na) calcd 591.4061, found 591.4061.

O N HO N O O O O OH

3-{5-[2-(2,3-Isopropylidenedioxyprop-1-yl)-m-carboran-1-yl]pentan-1-yl}thymidine

(3.10.D)

Compound 3.10.D was prepared from 3.9.D in 57% yield adapting the procedure

1 described for compound 3.10.A. Rf 0.25 (ethylacetate/methanol, 25:1); H-NMR

6 (Acetone-d ) δ 1.20-1.24 (m, 2H, CH2) 1.27 (s, 3H, CH3), 1.30 (s, 3H, CH3), 1.37-1.45

(m, 2H, CH2), 1.50-1.58 (m, 2H, CH2), 1.82 (s, 3H, CH3), 1.95-2.00 (m, 2H, H-2´), 2.21-

2.24 (m, 4H, CH2-Ccarborane), 3.44 (dd, 1H, CH2, J = 7.5, 5.8 Hz), 3.76-3.85 (m, 4H, CH2N

and H-5´), 3.92 (m, 1H, H-4´), 4.02-4.10 (m, 2H, CH2), 4.23 (t, 1H, OH-5´, J = 4.8 Hz),

4.39 (d, 1H, OH-3´, J = 3.9 Hz) 4.45-4.49 (m, 1H, H-3´), 6.32 (t, 1H, H-1´, J = 6.7 Hz),

13 6 7.81 (s, 1H, H-6); C-NMR (Acetone-d ) δ 13.28 (CH3), 25.76 (CH3), 26.93 (CH3),

27.10 (CH2), 27.75 (CH2), 30.32 (CH2), 37.38 (CH2-Ccarborane), 41.13 (CH2-Ccarborane),

41.89 (C-2´), 42.92 (CH2-N), 62.49 (C-5´), 69.38 (CH2), 71.78 (C-3´), 75.36 (CH), 86.19

(C-1´), 88.44 (C-4´), 109.59 (C-5), 109.62 [C(CH3)3], 135.26 (C-6), 151.48 (C-2), 163.52

+ (C-4); MS (HR-ESI) C23H44B10N2O7Na (M+Na) calcd 591.4061, found 591.4046.

140 O N HO N O O O OH O

3-{5-[2-(2,3-Isopropylidenedioxyprop-1-yl)-p-carboran-1-yl]pentan-1-yl}thymidine

(3.10.E)

Compound 3.10.E was prepared from 3.9.E in 62% yield adapting the procedure

1 described for compound 3.10.A. Rf 0.25 (ethylacetate/methanol, 25:1); H-NMR

4 (MeOH-d ) δ 1.10-1.20 (m, 4H, CH2) 1.25 (s, 3H, CH3), 1.27 (s, 3H, CH3), 1.47-1.53 (m,

2H, CH2), 1.60-1.64 (m, 2H, CH2-Ccarborane), 1.78-1.93 (m, 2H, CH2-Ccarborane), 1.89 (d,

3H, CH3, J = 1.2 Hz), 2.14-2.29 (m, 2H, H-2´), 3.30-3.32 (m, 1H, CH2), 3.70-3.95 (m,

7H, CH2N, H-5´, CH2, and CH), 4.36-4.40 (m, 1H, H-4´), 6.28 (t, 1H, H-1´, J = 6.7 Hz),

13 4 7.82 (d, 1H, H-6, J = 1.2 Hz); C-NMR (MeOH-d ) δ 13.20 (CH3), 25.66 (CH3), 27.16

(CH3), 27.32 (CH2), 28.01 (CH2), 30.29 (CH2), 38.73 (CH2-Ccarborane), 41.34 (CH2-

Ccarborane), 41.99 (C-2´), 42.96 (CH2-N), 62.76 (C-5´), 69.93 (CH2), 72.10 (C-3´), 75.89

(CH), 77.21 (C-Ccarborane), 81.43 (C-Ccarborane), 87.09 (C-1´), 88.87 (C-4´), 110.23 (C-5),

110.68 [C(CH3)3], 136.46 (C-6), 152.28 (C-2), 165.39 (C-4); MS (HR-ESI)

+ C23H44B10N2O7Na (M+Na) calcd 591.4061, found 591.4062.

N OH HO N O OH O

OH 141

(R)-3-{5-[2-(2,3-Dihydroxyprop-1-yl)-o-carboran-1-yl]pentan-1-yl}thymidine

(3.11.A)

To a solution of compound 3.10.A (40 mg, 0.07 mmol) in MeOH (5 mL) was

added a mixture of 3N-HCl and EtOH (1 mL, 1:1). The reaction mixture was stirred at

room temperature for 20 h. Potassium carbonate (17 mg) was added to the reaction

mixture, which was then stirred for 30 min at room temperature. The reaction mixture

was filtered using a Buchner funnel to remove the solids and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel columns chromatography using ethylacetate/methanol (15:1) as the eluent to give compound 3.11.A (30 mg, 81%).

25 1 4 Rf 0.25; [α]D +17.5 (c = 0.15, MeOH); H-NMR (MeOH-d ) δ 1.30-1.38 (m, 2H, CH2),

1.55-1.65 (m, 4H, CH2), 1.89 (d, 3H, CH3, J = 1.1 Hz), 2.15-2.39 (m, 5H, H-2´, CH2-

Ccarborane, and CH(OH)-CH2-Ccarborane), 2.56 (dd, 1H, CH2-Ccarborane, J = 15.8, 1.5 Hz), 3.34

(dd, 1H, CH2OH, J = 11.0, 6.5 Hz), 3.47 (dd, 1H, CH2OH, J = 11.0, 5.3 Hz), 3.72 (dd,

1H, H-5´, J = 12.1, 3.7 Hz), 3.75-3.79 (m, 1H, CH(OH)-CH2OH), 3.79 (dd, 1H, H-5´, J =

12.1, 3.2 Hz), 3.88-3.91 (m, 3H, H-4´ and CH2N), 4.37-4.40 (m, 1H, H-3´), 6.29 (t, 1H,

13 4 H-1´, J = 6.8 Hz), 7.83 (d, 1H, H-6, J = 1.1 Hz); C-NMR (MeOH-d ) δ 13.26 (CH3),

27.27 (CH2), 27.88 (CH2), 30.36 (CH2), 35.77 (CH2), 39.84 (CH2), 41.32 (CH2), 41.92

(CH2), 62.73 (O-CH2), 66.89 (O-CH2), 72.07 (O-CH2), 72.17 (O-CH2), 80.39 (Ccarborane-

C), 81.85 (Ccarborane-C), 87.06 (O-CH), 88.81 (O-CH), 110.67 (C-5), 136.44 (C-6), 152.26

+ (C-2), 165.37 (C-4); MS (HR-ESI) C20H40B10N2O7Na (M+Na) calcd 551.3747, found

551.3755; HPLC retention time: 26.7 min (Method A), 21.1 min (Method B).

142 O

N OH HO N O OH O

(S)-3-{5-[2-(2,3-Dihydroxyprop-1-yl)-o-carboran-1-yl]pentan-1-yl}thymidine

(3.11.B)

Compound 3.11.B was prepared from 3.10.B in 85% yield adapting the procedure

25 described for compound 3.11.A. Rf 0.25 (ethylacetate/methanol, 15:1); [α]D -14.8 (c =

1 4 0.1, MeOH); H-NMR (MeOH-d ) δ 1.30-1.38 (m, 2H, CH2), 1.54-1.65 (m, 4H, CH2),

1.89 (d, 3H, CH3, J = 1.1 Hz), 2.15-2.39 (m, 5H, H-2´, CH2- Ccarborane, and CH(OH)-CH2-

Ccarborane), 2.56 (dd, 1H, CH2-Ccarborane, J = 15.8, 1.6 Hz), 3.33 (dd, 1H, CH2OH, J = 11.0,

6.5 Hz), 3.47 (dd, 1H, CH2OH, J = 11.0, 5.3 Hz), 3.72 (dd, 1H, H-5´, J = 12.1, 3.7 Hz),

3.75-3.79 (m, 1H, CH(OH)-CH2OH), 3.79 (dd, 1H, H-5´, J = 12.1, 3.2 Hz), 3.88-3.92 (m,

3H, H-4´ and CH2N), 4.37-4.40 (m, 1H, H-3´), 6.29 (t, 1H, H-1´, J = 6.8 Hz), 7.83 (d, 1H,

13 4 H-6, J = 1.1 Hz); C-NMR (MeOH-d ) δ 13.24 (CH3), 27.28 (CH2), 27.90 (CH2), 30.37

(CH2), 35.79 (CH2), 39.87 (CH2), 41.33 (CH2), 41.92 (CH2), 62.75 (O-CH2), 66.90 (O-

CH2), 72.09 (O-CH2), 72.20 (O-CH2), 80.42 (Ccarborane-C), 81.87 (Ccarborane-C), 87.09 (O-

CH), 88.85 (O-CH), 110.69 (C-5), 136.46 (C-6), 152.30 (C-2), 165.41 (C-4); MS (HR-

+ ESI) C20H40B10N2O7Na (M+Na) calcd 551.3747, found 551.3749; HPLC retention time:

26.6 min (Method A), 21.1 min (Method B).

143 O

N OH HO N O OH O

3-{5-[2-(2,3-Dihydroxyprop-1-yl)-o-carboran-1-yl]pentan-1-yl}thymidine (3.11.C)

Compound 3.11.C was prepared from 3.10.C in 80% yield following the

1 procedure described for compound 3.11.A. Rf 0.25 (ethylacetate/methanol, 15:1); H-

4 NMR (MeOH-d ) δ 1.30-1.37 (m, 2H, CH2), 1.55-1.65 (m, 4H, CH2), 1.89 (d, 3H, CH3, J

= 1.1 Hz), 2.18-2.42 (m, 5H, H-2´, CH2-Ccarborane, and CH(OH)-CH2-Ccarborane), 2.55 (dd,

1H, CH2-Ccarborane, J = 15.8, 1.5 Hz), 3.33 (dd, 1H, CH2OH, J = 11.0, 6.5 Hz), 3.46 (dd,

1H, CH2OH, J = 11.0, 5.3 Hz), 3.71 (dd, 1H, H-5´, J = 12.1, 3.7 Hz), 3.74-3.79 (m, 1H,

CH(OH)-CH2OH), 3.79 (dd, 1H, H-5´, J = 12.1, 3.2 Hz), 3.88-3.92 (m, 3H, H-4´ and

CH2N), 4.37-4.40 (m, 1H, H-3´), 6.29 (t, 1H, H-1´, J = 6.8 Hz), 7.83 (d, 1H, H-6, J = 1.1

13 4 Hz); C-NMR (MeOH-d ) δ 13.22 (CH3), 27.29 (CH2), 27.90 (CH2), 30.38 (CH2), 35.81

(CH2), 39.90 (CH2), 41.33 (CH2), 41.92 (CH2), 62.76 (O-CH2), 66.91 (O-CH2), 72.12 (O-

CH2), 72.21 (O-CH2), 80.45 (Ccarborane-C), 81.90 (Ccarborane-C), 87.10 (O-CH), 88.88 (O-

CH), 110.71 (C-5), 136.48 (C-6), 152.33 (C-2), 165.44 (C-4); MS (HR-ESI)

+ C20H40B10N2O7Na (M+Na) calcd 551.3747, found 551.3753; HPLC retention time: 26.9 min (Method A), 21.0 min (Method B).

144 O N HO N O O OH OH OH

3-{5-[7-(2,3-Dihydroxyprop-1-yl)-m-carboran-1-yl]pentan-1-yl}thymidine (3.11.D)

Compound 3.11.D was prepared from 3.10.D in 88% yield following the

1 procedure described for compound 3.11.A. Rf 0.25 (ethylacetate/methanol, 15:1); H-

4 NMR (MeOH-d ) δ 1.21-1.28 (m, 2H, CH2), 1.38-1.44 (m, 2H, CH2), 1.53-1.60 (m, 2H,

CH2), 1.89 (d, 3H, CH3, J = 1.1 Hz), 1.92-1.98 (m, 2H, CH2-Ccarborane), 2.16-2.30 (m, 4H,

H-2´ and CH2-Ccarborane), 3.26 (dd, 1H, CH2OH, J = 11.1, 6.1 Hz), 3.36 (dd, 1H, CH2OH, J

= 11.1, 5.5 Hz), 3.55-3.60 (m, 1H, CH(OH)-CH2OH), 3.72 (dd, 1H, H-5´, J = 12.1, 3.7

Hz), 3.79 (dd, 1H, H-5´, J = 12.1, 3.2 Hz), 3.86-3.92 (m, 3H, H-4´ and CH2N), 4.37-4.40

(m, 1H, H-3´), 6.29 (t, 1H, H-1´, J = 6.8 Hz), 7.83 (d, 1H, H-6, J = 1.1 Hz); 13C-NMR

4 (MeOH-d ) δ 13.22 (CH3), 27.40 (CH2), 28.05 (CH2), 30.74 (CH2), 37.98 (CH2), 41.34

(CH2), 41.80 (CH2), 42.01 (CH2), 62.75 (O-CH2), 66.94 (O-CH2), 72.10 (O-CH2), 72.35

(O-CH2), 75.21 (Ccarborane-C), 77.68 (Ccarborane-C), 87.11 (O-CH), 88.86 (O-CH), 110.69

(C-5), 136.47 (C-6), 152.29 (C-2), 165.40 (C-4); MS (HR-ESI) C20H40B10N2O7Na

(M+Na)+ calcd 551.3747, found 551.3740; HPLC retention time: 25.7 min (Method A),

21.0 min (Method B).

145 O N HO N O O OH OH OH

3-{5-[12-(2,3-Dihydroxyprop-1-yl)-p-carboran-1-yl]pentan-1-yl}thymidine (3.11.E)

Compound 3.11.E was prepared from 3.10.E in 88% yield following the

1 procedure described for compound 3.11.A. Rf 0.25 (ethylacetate/methanol, 15:1); H-

4 NMR (MeOH-d ) δ 1.10-1.20 (m, 4H, CH2), 1.47-1.53 (m, 2H, CH2), 1.60-1.66 (m, 3H,

CH2-Ccarborane), 1.89 (d, 3H, CH3, J = 1.1 Hz), 1.89-1.92 (m, 1H, CH2-Ccarborane), 2.14-2.29

(m, 2H, H-2´), 3.16 (dd, 1H, CH2OH, J = 11.0, 6.0 Hz), 3.24 (dd, 1H, CH2OH, J = 11.0,

5.6 Hz), 3.32-3.38 (m, 1H, CH), 3.71 (dd, 1H, H-5´, J = 12.1, 3.7 Hz), 3.79 (dd, 1H,

CH2OH, J = 12.1, 3.1 Hz), 3.83 (t, 2H, CH2N, J = 7.6 Hz), 3.88-3.91 (m, 1H, H-4´), 4.36-

4.40 (m, 1H, H-3´), 6.28 (t, 1H, H-1´, J = 6.8 Hz), 7.82 (d, 1H, H-6, J = 1.1 Hz); 13C-

4 NMR (MeOH-d ) δ 13.17 (CH3), 27.33 (CH2), 28.01 (CH2), 30.27 (CH2), 38.71 (CH2),

41.34 (CH2), 42.00 (CH2), 42.52 (CH2), 62.76 (O-CH2), 66.87 (O-CH2), 72.07 (O-CH2),

72.16 (O-CH2), 78.74 (Ccarborane-C), 81.23 (Ccarborane-C), 87.12 (O-CH), 88.86 (O-CH),

110.69 (C-5), 136.43 (C-6), 152.28 (C-2), 165.38 (C-4); MS (HR-ESI) C20H40B10N2O7Na

(M+Na)+ calcd 551.3747, found 551.3746; HPLC retention time: 23.7 min (Method A),

19.9 min (Method B).

Iodination of N5-2OH (3.12.A-C)

146 To a solution of compound 3.5.A (90 mg, 0.17 mmol) in dichloromethane (15 mL) was added I2 (70 mg, 0.26 mmol) and AlCl3 (4.5 mg, 0.034 mmol). The reaction

mixture was stirred at 40 °C for 48 h. Aqueous solution of Na2S2O3 (70 mg) was added

to the reaction mixture and stirred at room temperature for additional 30 min.

Ethylacetate (15 mL) was added and the organic layer was separated, washed with brine,

and dried over magnesium sulfate. After filtration and evaporation, the residue was

separated by silica gel column chromatography using ethylacetate and methanol (15:1) as

the eluent, followed by separation of three products on the RP-18 analytical column using

the method A.

O N OH HO N O OH O I OH

3-{5-[2-(2,3-Dihydroxyprop-1-yl)-9-iodo-o-carboran-1-yl]pentan-1-yl}thymidine

(3.12.A)

1 Yield: 7 %; HPLC retention time: 26.5 min (Method A); H-NMR (CD3OD) δ

1.34-1.40 (m, 2H, CH2), 1.58-1.63 (m, 4H, CH2), 1.88 (d, 3H, CH3, J = 1.2 Hz), 2.12-2.37

(m, 5H, H-2´ and CH2-Ccarborane, and CH(OH)-CH2-Ccarborane), 2.46 (dd, 1H, CH(OH)-

CH2-Ccarborane, J = 15.8, 1.7 Hz), 3.31 (dd, 1H, CH2OH, J = 11.0, 6.5 Hz) 3.46 (dd, 1H,

CH2OH, J = 11.0, 5.3 Hz), 3.67-3.69 (m, 1H, CH(OH)-CH2OH), 3.71 (dd, 1H, H-5´, J =

12.0, 3.7 Hz), 3.79 (dd, 1H, H-5´, J = 12.0, 3.2 Hz), 3.89-3.93 (m, 3H, H-4´ and CH2N),

4.37-4.40 (m, 1H, H-3´), 6.30 (t, 1H, H-1´, J = 6.9 Hz), 7.83 (d, 1H, H-6, J = 1.2 Hz); 147 11 + B-NMR (CD3OD) δ -14.8, -9.9, -6.3; MS (HR-ESI) C20H39B10IN2O7Na (M+Na) calcd

677.2714, found 677.2759.

O N OH HO N O OH O I I OH

3-{5-[2-(2,3-Dihydroxyprop-1-yl)-9,12-diiodo-o-carboran-1-yl]pentan-1-

yl}thymidine (3.12.B)

1 Yield: 16 %; HPLC retention time 28.9 min (Method A); H-NMR (CD3OD) δ

1.32-1.40 (m, 2H, CH2), 1.56-1.66 (m, 4H, CH2), 1.89 (d, 3H, CH3, J = 1.2 Hz), 2.14-2.37

(m, 5H, H-2´ and CH2-Ccarborane, and CH(OH)-CH2-Ccarborane), 2.52 (dd, 1H, CH(OH)-

CH2-Ccarborane, J = 15.8, 1.7 Hz), 3.31 (dd, 1H, CH2OH, J = 11.0, 6.5 Hz) 3.48 (dd, 1H,

CH2OH, J = 11.0, 5.3 Hz), 3.71-3.74 (m, 1H, CH(OH)-CH2OH), 3.72 (dd, 1H, H-5´, J =

12.0, 3.7 Hz), 3.79 (dd, 1H, H-5´, J = 12.0, 3.2 Hz), 3.89-3.92 (m, 3H, H-4´ and CH2N),

4.37-4.40 (m, 1H, H-3´), 6.30 (t, 1H, H-1´, J = 6.9 Hz), 7.83 (d, 1H, H-6, J = 1.2 Hz);

13 C-NMR (CD3OD) δ 13.21 (CH3), 27.12 (CH2), 27.78 (CH2), 30.31 (CH2), 35.32 (C-

Ccarborane), 39.49 (C-Ccarborane), 41.34 (C-2´), 41.83 (CH2N), 62.78 (C-5´), 66.83 (O-CH2),

72.00 (O-CH), 72.13 (C-3´), 78.37 (Ccarborane-C), 79.66 (Ccarborane-C), 87.13 (C-1´), 88.89

11 (C-4´), 110.73 (C-5), 136.50 (C-6), 152.35 (C-2), 165.44 (C-4); B-NMR (CD3OD) δ -

+ 12.6, -6.6, -4.0; MS (HR-ESI) C20H38B10I2N2O7Na (M+Na) calcd 803.1680, found

803.1683.

148

O N OH HO N O OH O I I I OH

3-{5-[2-(2,3-Dihydroxyprop-1-yl)-9,11,12-triiodo-o-carboran-1-yl]pentan-1-

yl}thymidine (3.12.C)

1 Yield: 10 %; HPLC retention time 30.62 min (Method A); H-NMR (CD3OD) δ

1.35-1.39 (m, 2H, CH2), 1.57-1.65 (m, 4H, CH2), 1.89 (d, 3H, CH3, J = 1.2 Hz), 2.17-2.33

(m, 5H, H-2´ and CH2-Ccarborane, and CH(OH)-CH2-Ccarborane), 2.54 (dd, 1H, CH(OH)-

CH2-Ccarborane, J = 15.8, 1.7 Hz), 3.33 (dd, 1H, CH2OH, J = 10.9, 6.4 Hz) 3.49 (dd, 1H,

CH2OH, J = 10.9, 5.2 Hz), 3.72 (dd, 1H, H-5´, J = 12.0, 3.7 Hz), 3.72-3.74 (m, 1H,

CH(OH)-CH2OH), 3.79 (dd, 1H, H-5´, J = 12.0, 3.2 Hz), 3.89-3.93 (m, 3H, H-4´ and

CH2N), 4.37-4.40 (m, 1H, H-3´), 6.30 (t, 1H, H-1´, J = 6.9 Hz), 7.83 (d, 1H, H-6, J = 1.2

11 Hz); B-NMR (CD3OD) δ -15.0, -10.0, -6.2; MS (HR-ESI) C20H37B10I3N2O7Na

(M+Na)+ calcd 929.0646, found 929.0649.

7.3. Chapter 4

H H N O

149 1-{[(tert-Butyloxy)carbonyl]amino}-1,7-closo-m-carborane (4.1)

Compound 4.1 was synthesized according to a procedure reported by Karl and

121 1 coworkers. H-NMR (CDCl3) δ 1.46 (s, 9H, t-butyl), 2.93 (br s, 1H, H-Ccarborane), 5.17

+ (br s, 1H, NH); MS (HR-ESI) C7H21B10NO2Na (M+Na) calcd: 282.2468, found:

282.2478.

H O N O S O O O

2-{[7-(tert-Butyloxy)carbonylamino]-closo-m-carboran-1-yl}ethyl tosylate (4.2)

n-BuLi in hexanes (2.5 M, 1.53 mL, 3.83 mmol) was slowly added to a solution

of compound 4.1 (450 mg, 1.74 mmol) in benzene (15 mL) at 5-10 °C and stirred for 20

min at the same temperature. The mixture was warmed to room temperature and stirred

for additional 30 min. The solution was added very slowly to a solution of ethylene

glycol di-p-tosylate (1.6 g, 4.32 mmol) in benzene (5 mL) at 5-10 °C. The mixture was

stirred for 30 min at the same temperature, distilled water (20 mL) was added, and benzene was removed under reduced pressure. The residue was extracted with ethyl acetate (15 mL × 3) and the combined organic layers were washed with brine, and dried over magnesium sulfate. After filtration and evaporation, the residue was purified by

silica gel column chromatography (hexanes/ethylacetate, 12:1) to give compound 4.2 as

1 colorless oil in 35% (280 mg) yield. Rf 0.15; H-NMR (CDCl3) δ 1.40 (s, 9H, C(CH3)3),

2.29 (t, 2H, CH2-Ccarborane, J = 7.1 Hz), 2.44 (s, 3H, CH3), 3.94 (t, 2H, CH2O, J = 7.1 Hz),

150 5.07 (s, 1H, NH), 7.34 (d, 2H, Ar-H, J = 8.3 Hz ), 7.76 (m, 2H, Ar-H, J = 8.3 Hz ); 13C

NMR (CDCl3) δ 21.69 (CH3), 28.13 (CH3), 33.47 (CH2-Ccarborane), 67.35 [OC(CH3)3],

69.37 (CH2O), 80.72 (Ccarborane-C), 82.89 (Ccarborane-C), 127.93 (Ar-C), 129.98 (Ar-C),

+ 132.97 (Ar-C), 145.05 (Ar-C), 152.04 (C=O); MS (HR-ESI) C16H31B10NO5SNa (M+Na) calcd: 480.2833, found: 480.2802.

O O H S N O O O

3-{[7-(tert-Butyloxy)carbonylamino]-closo-m-carboran-1-yl}propyl tosylate (4.3)

Compound 4.3 was prepared according to the procedure described for compound

4.2 using n-BuLi in hexanes (2.5 M, 1.04 mL, 2.60 mmol), compound 4.1 (307 mg, 1.18 mmol), and 1,3-propanediol di-p-tosylate (1.00 g, 2.6 mmol). Purification by silica gel column chromatography (hexanes/ethylacetate, 12:1) gave 4.3 as colorless oil in 36%

1 (200 mg) yield. Rf 0.16; H-NMR (CDCl3) δ 1.40 (s, 9H, C(CH3)3), 1.63-1.67 (m, 2H,

CH2), 1.90-1.97 (m, 2H, CH2-Ccarborane), 2.43 (s, 3H, CH3), 3.91 (t, 2H, CH2O, J = 7.0

Hz), 5.13 (s, 1H, NH), 7.33 (d, 2H, Ar-H, J = 8.1 Hz), 7.73 (d, 2H, Ar-H, J = 8.1 Hz); 13C

NMR (CDCl3) δ 21.65 (CH3), 28.14 (CH3), 28.99 (CH2), 33.03 (CH2-Ccarborane), 69.01

[OC(CH3)3], 72.37 (CH2O), 80.72 (Ccarborane-C), 81.70 (Ccarborane-C), 127.89 (Ar-C),

129.96 (Ar-C), 132.78 (Ar-C), 145.04 (Ar-C), 151.85 (C=O); MS (HR-ESI)

+ C17H33B10NO5SNa (M+Na) calcd: 494.2975, found: 494.2997.

151 H O N O S O O O

4-{[7-(tert-Butyloxy)carbonylamino]-closo-m-carboran-1-yl}butyl tosylate (4.4)

Compound 4.4 was prepared according to the procedure described for compound

4.2 using n-BuLi in hexanes (2.5 M, 1.00 mL, 2.50 mmol), compound 4.1 (258 mg, 1.00

mmol), and 1,4-butanediol di-p-tosylate (600 mg, 1.51 mmol). Purification by silica gel

column chromatography (hexanes/ethylacetate, 14:1) gave 8 as colorless oil in 43% (210

1 mg) yield. Rf 0.14; H-NMR (CDCl3) δ 1.25-1.35 (m, 4H, CH2), 1.41 (s, 9H, C(CH3)3),

1.49-1.52 (m, 2H, CH2), 1.81-1.88 (m, 2H, CH2-Ccarborane), 2.44 (s, 3H, CH3), 3.95 (t, 2H,

CH2O, J = 6.2 Hz), 5.09 (s, 1H, NH), 7.34 (d, 2H, Ar-H, J = 8.0 Hz), 7.77 (d, 2H, Ar-H, J

13 = 8.0 Hz); C NMR (CDCl3) δ 21.64 (CH3), 25.81 (CH2), 28.12 (CH3), 28.25 (CH2),

36.31 (CH2-Ccarborane), 69.65 [OC(CH3)3], 73.21 (CH2O), 80.51 (Ccarborane-C), 81.63

(Ccarborane-C), 127.86 (Ar-C), 129.93 (Ar-C), 132.87 (Ar-C), 144.89 (Ar-C), 151.86

+ (C=O); MS (HR-ESI) C18H35B10NO5SNa (M+Na) calcd: 508.3147, found: 508.3169.

O O H S N O O O

5-{[7-(tert-Butyloxy)carbonylamino]-closo-m-carboran-1-yl}pentyl tosylate (4.5)

Compound 4.5 was prepared according to the procedure described for compound

4.2 using n-BuLi in hexanes (2.5 M, 1.82 mL, 4.56 mmol), compound 4.1 (560 mg, 2.17

152 mmol), and 1,5-pentanediol di-p-tosylate (983 mg, 2.39 mmol). Purification by silica gel

column chromatography (hexanes/ethylacetate, 14:1) gave 4.5 as colorless oil in 39%

1 (420 mg) yield. Rf 0.16; H-NMR (CDCl3) δ 1.20-1.28 (m, 4H, CH2), 1.42 (s, 9H,

C(CH3)3), 1.57-1.62 (m, 2H, CH2), 1.83-1.89 (m, 2H, CH2-Ccarborane), 2.45 (s, 3H, CH3),

3.98 (t, 2H, CH2O, J = 6.2 Hz), 5.12 (s, 1H, NH), 7.34 (d, 2H, Ar-H, J = 7.9 Hz), 7.78 (d,

13 2H, Ar-H, J = 7.9 Hz); C-NMR (CDCl3) δ 21.63 (CH3), 24.88 (CH2), 28.12 (CH3),

28.35 (CH2), 29.10 (CH2), 36.76 (CH2-Ccarborane), 70.03 [OC(CH3)3], 73.60 (CH2O), 80.52

(Ccarborane-C), 81.59 (Ccarborane-C), 127.84 (Ar-C), 129.85 (Ar-C), 133.02 (Ar-C), 144.79

+ (Ar-C), 151.89 (C=O); MS (HR-ESI) C19H37B10NO5SNa (M+Na) calcd: 522.3288,

found: 522.3263.

H O N O S O O O

6-{[7-(tert-Butyloxy)carbonylamino]-closo-m-carboran-1-yl}hexyl tosylate (4.6)

Compound 4.6 was prepared according to the procedure described for compound

4.2 using n-BuLi in hexanes (2.5 M, 1.00 mL, 2.50 mmol), compound 4.1 (258 mg, 1.00

mmol), and 1,6-hexanediol di-p-tosylate (650 mg, 1.52 mmol). Purification by silica gel

column chromatography (hexanes/ethylacetate, 15:1) gave 4.6 as colorless oil in 45%

1 (230 mg) yield. Rf 0.16; H-NMR (CDCl3) δ 1.10-1.12 (m, 2H, CH2), 1.21-1.27 (m, 4H,

CH2), 1.40 (s, 9H, C(CH3)3), 1.55-1.59 (m, 2H, CH2), 1.82-1.86 (m, 2H, CH2-Ccarborane),

2.43 (s, 3H, CH3), 3.97 (t, 2H, CH2O, J = 6.4 Hz), 5.11 (s, 1H, NH), 7.32 (d, 2H, Ar-H, J

153 13 = 8.0 Hz), 7.74 (d, 2H, Ar-H, J = 8.0 Hz); C-NMR (CDCl3) δ 21.63 (CH3), 24.95

(CH2), 28.12 (CH3), 28.36 (CH2), 28.58 (CH2), 29.62 (CH2), 36.88 (CH2-Ccarborane), 70.31

[OC(CH3)3], 73.85 (CH2O), 80.49 (Ccarborane-C), 81.57 (Ccarborane-C), 127.85 (Ar-C),

129.81(Ar-C), 133.08 (Ar-C), 144.70 (Ar-C), 151.89 (C=O); MS (HR-ESI)

+ C20H39B10NO5SNa (M+Na) calcd: 536.3461, found: 536.3467.

O O H S O N O O O

2-{2-[7-(tert-Butyloxy)carbonylamino]-closo-m-carboran-1-yl}ethyloxyethyl tosylate

(4.7)

Compound 4.7 was prepared according to the procedure described for compound

4.2 using n-BuLi in hexanes (2.5 M, 2.04 mL, 5.10 mmol), compound 4.1 (600 mg, 2.32

mmol), and di(ethylene glycol) di-p-tosylate (1.05 g, 2.55 mmol). Purification by silica gel column chromatography (hexanes/ethylacetate, 5:1) gave 4.7 as colorless oil in 14%

1 (160 mg) yield. Rf 0.19, H-NMR (CDCl3): δ 1.40 (s, 9H, C(CH3)3), 2.09 (t, 2H, CH2-

Ccarborane, J = 6.8 Hz), 2.44 (s, 3H, CH3), 3.30 (t, 2H, CH2O, J = 6.8 Hz), 3.53 (t, 2H,

CH2O, J = 4.5 Hz), 4.11 (t, 2H, CH2O, J = 4.5 Hz), 5.13 (br s, 1H, NH), 7.32 (d, 2H, Ar-

H, J = 7.9 Hz), 7.77 (d, 2H, Ar-H, J = 7.9 Hz); MS (HR-ESI) C18H35B10NO6SNa

(M+Na)+ calcd: 524.3086, found: 524.3257.

154 H O N O N O HO N O O

3-{2-[(7-(tert-Butyloxy)carbonylamino)-closo-m-carboran-1-yl]ethan-1-yl}thymidine

(4.8)

A solution of compound 4.2 (270 mg, 0.59 mmol), dThd (440 mg, 1.82 mmol),

potassium carbonate (450 mg, 3.26 mmol) in DMF/acetone (20 mL, 1:1) was stirred at 50

°C for 48 h. The solution was filtered and evaporated. The residue was dissolved in the

water (20 mL) and extracted with ethyl acetate (20 mL × 4). The combined organic layers

were washed with brine and dried over magnesium sulfate. After filtration and evaporation, the residue was purified by silica gel column chromatography (ethyl

acetate/methanol, 15:1) to give compound 4.8 as a white solid in 64% (200 mg) yield. Rf

1 0.15; H-NMR (CDCl3) δ 1.39 (s, 9H, C(CH3)3), 1.86 (s, 3H, CH3), 2.15-2.20 (m, 2H, H-

2´), 2.27-2.32 (t, 2H, CH2-Ccarborane, J = 6.3 Hz), 3.82-3.99 (m, 5H, H-4´, H-5´ and CH2N),

4.51-4.55 (m, 1H, H-3´), 5.34 (s, 1H, NH), 6.17 (t, 1H, H-1´, J = 6.7 Hz), 7.43 (s, 1H, H-

13 4 6); C-NMR (MeOH-d ) δ 13.09 (CH3), 28.51 (CH3), 34.21 (CH2-Ccarborane), 41.31

(CH2N), 41.39 (C-2´), 62.69 (C-5´), 71.45 (C-3´), 72.01 [OC(CH3)3], 81.66 (Ccarborane-C),

83.21 (Ccarborane-C), 87.12 (C-1´), 88.85 (C-4´), 110.60 (C-5), 136.65 (C-6), 151.86

+ (C=O), 154.58 (C=O), 164.92 (C=O); MS (HR-ESI) C19H37B10N3O7Na (M+Na) calcd:

550.3542, found: 550.3578.

155 O H N N O HO N O O O

3-{3-[(7-(tert-Butyloxy)carbonylamino)-closo-m-carboran-1-yl]propan-1-

yl}thymidine (4.9).

Compound 4.9 was prepared according to the procedure described for compound

4.8 using compound 4.3 (170 mg, 0.36 mmol), dThd (104 mg, 0.43 mmol), potassium

carbonate (150 mg, 1.08 mmol). Purification by silica gel column chromatography (ethyl

1 acetate/methanol, 15:1) gave 4.9 as a white solid in 85% (166 mg) yield. Rf 0.17; H-

NMR (CDCl3) δ 1.40 (s, 9H, C(CH3)3), 1.56-1.67 (m, 2H, CH2), 1.90 (s, 3H, CH3), 1.90-

2.02 (m, 2H, H-2´), 2.30-2.38 (m, 2H, CH2-Ccarborane), 3.80-3.98 (m, 5H, H-4´, H-5´ and

CH2N), 4.56-4.59 (m, 1H, H-3´), 5.15 (s, 1H, NH), 6.17 (t, 1H, H-1´), 7.33 (s, 1H, H-6);

13 4 C-NMR (MeOH-d ) δ 13.22 (CH3), 28.53 (CH2), 29.10 (CH3), 36.53 (CH2-Ccarborane),

41.32 (CH2N), 41.55 (C-2´), 62.72 (C-5´), 72.04 [OC(CH3)3], 74.09 (C-3´), 81.62

(Ccarborane-C), 82.99 (Ccarborane-C), 87.10 (C-1´), 88.84 (C-4´), 110.68 (C-5), 136.61 (C-6),

+ 152.22 (C=O), 154.59 (C=O), 163.31 (C=O); MS (HR-ESI) C20H39B10N3O7Na (M+Na) calcd: 564.3616, found: 564.3638.

H O N O N O HO N O O

OH 156

3-{4-[(7-(tert-Butyloxy)carbonylamino)-closo-m-carboran-1-yl]butan-1-yl}thymidine

(4.10).

Compound 4.10 was prepared according to the procedure described for compound

4.8 using compound 4.4 (206 mg, 0.42 mmol), dThd (252 mg, 1.04 mmol), potassium

carbonate (275 mg, 1.99 mmol). Purification by silica gel column chromatography

(dichloromethane/acetone, 2:1) gave 14 as a white solid in 69% (160 mg) yield. Rf 0.19;

1 H-NMR (CDCl3) δ 1.35-1.38 (m, 2H, CH2), 1.40 (s, 9H, C(CH3)3), 1.48-1.50 (m, 2H,

CH2), 1.90 (d, 3H, CH3, J = 1.1 Hz), 1.92-1.97 (m, 2H, CH2-Ccarborane), 2.30-2.46 (m, 2H,

H-2´), 3.80-3.94 (m, 4H, H-5´ and CH2N), 3.98-4.00 (m, 1H, H-4´), 4.59-4.61 (m, 1H, H-

13 3´), 6.11 (t, 1H, H-1´, J = 6.8 Hz), 7.29 (d, 1H, H-6, J = 1.1 Hz); C-NMR (CDCl3) δ

13.27 (CH3), 26.87 (CH2), 27.09 (CH2), 28.14 (CH3), 36.47 (CH2-Ccarborane), 40.11

(CH2N), 40.55 (C-2´), 62.45 (C-5´), 71.57 [OC(CH3)3], 71.84 (C-3´), 80.40 (Ccarborane-C),

81.53 (Ccarborane-C), 86.81 (C-1´), 88.00 (C-4´), 110.35 (C-5), 135.05 (C-6), 150.84

+ (C=O), 151.97 (C=O), 163.17 (C=O); MS (HR-ESI) C21H41B10N3O7Na (M+Na) calcd:

578.3773, found: 578.3887.

O H N N O HO N O O O

157 3-{5-[(7-(tert-Butyloxy)carbonylamino)-closo-m-carboran-1-yl]pentan-1-

yl}thymidine (4.11).

Compound 4.11 was prepared according to the procedure described for compound

4.8 using compound 4.5 (380 mg, 0.76 mmol), dThd (460 mg, 1.9 mmol), potassium

carbonate (420 mg, 3.04 mmol). Purification by silica gel column chromatography

(dichloromethane/acetone, 2:1) gave 4.11 as a white solid in 79% (340 mg) yield. Rf

1 6 0.21; H-NMR (acetone-d ) δ 1.23-1.32 (m, 2H, CH2), 1.36 (s, 9H, C(CH3)3), 1.39-1.45

(m, 2H, CH2), 1.52-1.61 (m, 2H, CH2), 1.81-1.82 (d, 3H, CH3, J = 1.2 Hz), 2.02-2.07 (m,

2H, CH2-Ccarborane), 2.20-2.25 (m, 2H, H-2´), 3.75-3.79 (m, 2H, H-5´), 3.81-3.87 (m, 2H,

CH2N), 3.90-3.94 (m, 1H, H-4´), 4.21-4.26 (t, 1H, OH-5´, J = 5.0 Hz), 4.38-4.40 (d, 1H,

OH-3´, J = 4.2 Hz), 4.44-4.51 (m, 1H, H-3´), 6.33 (t, 1H, H-1´, J = 6.6 Hz), 7.49 (br s,

13 1H, NH), 7.81 (q, 1H, H-6, J = 1.2 Hz); C-NMR (CDCl3) δ 13.03 (CH3), 26.24 (CH2),

26.94 (CH2), 27.94 (CH3), 31.24 (CH2), 36.34 (CH2-Ccarborane), 40.21 (CH2N), 40.85 (C-

2´), 61.98 (C-5´), 70.97 [OC(CH3)3], 73.65 (C-3´), 80.56 (Ccarborane-C), 81.11 (Ccarborane-

C), 86.37 (C-1´), 86.94 (C-4´), 109.81 (C-5), 134.55 (C-6), 150.70 (C=O), 151.98 (C=O),

+ 163.27 (C=O); MS (HR-ESI) C22H43B10N3O7Na (M+Na) calcd: 592.3999, found:

592.3986.

H O N O N O HO N O O

158 3-{6-[(7-(tert-Butyloxy)carbonylamino)-closo-m-carboran-1-yl]hexan-1-yl}thymidine

(4.12).

Compound 4.12 was prepared according to the procedure described for compound

4.8 using compound 4.6 (208 mg, 0.40 mmol), dThd (262 mg, 1.08 mmol), potassium

carbonate (273 mg, 1.98 mmol). Purification by silica gel column chromatography

(dichloromethane/acetone, 2:1) gave 4.12 as a white solid in 55% (128 mg) yield. Rf

1 0.23; H-NMR (CDCl3) δ 1.18-1.29 (m, 6H, CH2), 1.39 (s, 9H, C(CH3)3), 1.52-1.54 (m,

2H, CH2), 1.86-1.89 (m, 2H, CH2-Ccarborane ), 1.90 (d, 3H, CH3, J = 1.0 Hz), 2.30-2.40 (m,

2H, H-2´), 3.79-3.92 (m, 4H, H-5´ and CH2N), 3.97-4.00 (m, 1H, H-4´), 4.55-4.59 (m,

1H, H-3´), 6.15 (t, 1H, H-1´, J = 6.8 Hz), 7.32 (d, 1H, H-6, J = 1.1 Hz); 13C-NMR

(CDCl3) δ 13.28 (CH3), 26.37 (CH2), 27.26 (CH2), 28.13 (CH3), 28.67 (CH2), 29.71

(CH2), 36.96 (CH2-Ccarborane), 40.08 (CH2N), 41.15 (C-2´), 62.40 (C-5´), 71.52

[OC(CH3)3], 74.03 (C-3´), 80.45 (Ccarborane-C), 81.59 (Ccarborane-C), 86.86 (C-1´), 87.65

(C-4´), 110.33 (C-5), 134.86 (C-6), 150.95 (C=O), 152.02 (C=O), 163.30 (C=O); MS

+ (HR-ESI) C23H45B10N3O7Na (M+Na) calcd: 606.4085, found: 606.4249.

O H O N N O HO N O O O

3-{2-[(7-(tert-Butyloxy)carbonylamino)-closo-m-carboran-1-yl]ethyloxyethyl}

thymidine (4.13).

159 Compound 4.13 was prepared according to procedure described for compound 4.8

using compound 4.7 (160 mg, 0.32 mmol), dThd (200 mg, 0.80 mmol), potassium

carbonate (175 mg, 1.28 mmol). Purification by silica gel column chromatography (ethyl

1 acetate/methanol, 15:1) gave 4.13 as a white solid in 50% (90 mg) yield. Rf 0.15; H-

6 NMR (acetone-d ) δ 1.38 (s, 9H, C(CH3)3), 1.83 (d, 3H, CH3, J = 1.2 Hz), 2.15-2.20 (m,

2H, CH2-Ccarborane), 2.22-2.26 (m, 2H, H-2´), 3.40 (t, 2H, OCH2, J = 6.3 Hz), 3.57 (t, 2H,

OCH2, J = 6.0 Hz), 3.75-3.81 (m, 2H, H-5´), 3.94-3.96 (m, 1H, H-4´), 4.08 (t, 2H, CH2N,

J = 6.0 Hz), 4.26 (t, 1H, OH-5´, J = 5.1 Hz), 4.41 (d, 1H, OH-3´, J = 4.2 Hz), 4.47-4.50

(m, 1H, H-3´), 6.32-6.37 (t, 1H, H-1´, J = 6.9 Hz), 7.47 (br s, 1H, NH), 7.82-7.83 (d, 1H,

13 4 H-6, J = 1.2 Hz); C-NMR (MeOH-d ) δ 13.30 (CH3), 28.27 (CH3), 37.41 (CH2-

Ccarborane), 40.40 (CH2N), 41.15 (C-2´), 62.74 (C-5´), 67.45 (CH2), 69.37 (CH2), 71.80

[OC(CH3)3], 72.02 (C-3´), 79.45 (Ccarborane-C), 80.79 (Ccarborane-C), 86.40 (C-1´), 88.54

(C-4´), 109.79 (C-5), 135.62 (C-6), 151.67 (C=O), 153.26 (C=O) 163.75 (C=O); MS

+ (HR-ESI) C21H41B10N3O8Na (M+Na) calcd: 594.3788, found: 594.3777.

O NH2 N HO N O O

3-[2-(7-Amino-closo-m-carboran-1-yl)-ethan-1-yl]thymidine (4.14.A).

Trifluoroacetic acid (0.5 mL, 0.74 mmol) was added at to a solution of compound

4.8 (195 mg, 0.37 mmol) dissolved in dichloromethane (10 mL) 0 °C. The mixture was

160 stirred at room temperature for 24 h. After evaporation, triethylamine (1 mL) and distilled

water (10 mL) were added. (Caution: It is necessary to remove CF3COOH completely by

evaporation to avoid the generation of toxic fumes due to acid-base reaction). The residue was extracted with ethyl acetate (20 mL × 5). The combined organic layers were washed with brine, and dried over magnesium sulfate. After filtration and evaporation,

the residue was purified by silica gel column chromatography (ethyl acetate/MeOH, 15:1)

1 to give compound 4.14.A as a white solid in 85% (135 mg) yield. Rf 0.13; H-NMR

4 (MeOH-d ) δ 1.87 (d, 3H, CH3, J = 1.2 Hz), 2.11-2.29 (m, 4H, CH2-Ccarborane and H-2´),

3.72 (dd, 1H, H-5´, J = 12.0, 3.1 Hz), 3.80 (dd, 1H, H-5´, J = 12.0, 3.1 Hz), 3.86-3.93 (m,

3H, H-4´ and CH2N), 4.34-4.38 (m, 1H, H-3´), 6.25 (t, 1H, H-1´, J = 6.5 Hz), 7.81 (d, 1H,

13 4 H-6, J = 1.2 Hz); C-NMR (MeOH-d ) δ 13.08 (CH3), 34.03 (CH2-Ccarborane), 41.31

(CH2N), 41.45 (C-2´), 62.71 (C-5´), 72.04 (C-3´), 74.68 (Ccarborane-C), 87.13 (C-1´), 88.88

(C-4´), 89.93 (Ccarborane-C), 110.61 (C-5), 136.66 (C-6), 151.90 (C-2), 164.95 (C-4); MS

+ (HR-ESI) C14H29B10N3O5Na (M+Na) calcd: 450.3016, found: 450.3022; HPLC

retention time: 19.01 min (Method C), 18.47 min (Method D)

N NH2 HO N O O

3-[3-(7-Amino-closo-m-carboran-1-yl)propan-1-yl]thymidine (4.15.A).

161 Compound 4.15.A was prepared according to the procedure described for compound 4.14.A using compound 4.9 (1.10 g, 2.03 mmol) and trifluoroacetic acid (2.74 mL, 4.06 mmol). Purification by silica gel column chromatography (ethyl

1 acetate/methanol, 15:1) gave 4.15.A as a white solid in 87% (780 mg) yield. Rf 0.16; H-

4 NMR (MeOH-d ) δ 1.60-1.73 (m, 2H, CH3), 1.90 (s, 3H, CH3, J = 1.1 Hz), 1.95-2.02 (m,

2H, CH2-Ccarborane), 2.13-2.32 (m, 2H, H-2´), 3.69-3.84 (m, 4H, H-5´ and CH2N), 3.88-

3.93 (m, 1H, H-4´), 4.36-4.41 (m, 1H, H-3´), 6.29 (t, 1H, H-1´, J = 6.6 Hz), 7.82 (d, 1H,

13 4 H-6, J = 1.1 Hz); C-NMR (MeOH-d ) δ 13.16 (CH3), 29.16 (CH2), 35.30 (CH2-

Ccarborane), 41.31 (CH2N), 41.44 (C-2´), 62.74 (C-5´), 72.06 (C-3´), 74.71 (Ccarborane-C),

87.12 (C-1´), 88.87 (C-4´), 91.18 (Ccarborane-C), 110.68 (C-5), 136.62 (C-6), 152.24 (C-2),

+ 165.31 (C-4); MS (HR-ESI) C15H31B10N3O5Na (M+Na) calcd 464.3178, found

464.3184; HPLC retention time: 19.08 min (Method C), 18.54 min (Method D).

O NH2 N HO N O O

3-[4-(7-Amino-closo-m-carboran-1-yl)butan-1-yl]thymidine (4.16.A).

Compound 4.16.A was prepared according to the procedure described for compound 4.14.A using compound 4.10 (95 mg, 0.16 mmol) and trifluoroacetic acid

(0.22 mL, 0.32 mmol). Purification by silica gel column chromatography (ethyl

1 acetate/methanol, 15:1) gave 4.16.A as a white solid in 69% (48 mg) yield. Rf 0.18; H-

162 4 NMR (MeOH-d ) δ 1.34-1.39 (m, 2H, CH2), 1.49-1.55 (m, 2H, CH2), 1.89-1.90 (d, 3H,

CH3, J = 1.1 Hz), 1.96-2.01 (m, 2H, CH2-Ccarborane), 2.15-2.29 (m, 2H, H-2´), 3.72 (dd,

1H, H-5´, J = 12.1, 3.4 Hz), 3.79 (dd, 1H, H-5´, J = 12.1, 3.4 Hz), 3.85-3.91 (m, 3H,

CH2N and H-4´), 4.37-4.39 (m, 1H, H-3´), 6.29 (t, 1H, H-1´, J = 6.4 Hz), 7.84 (d, 1H, H-

13 4 6, J = 1.1 Hz); C-NMR (MeOH-d ) δ 13.21 (CH3), 27.99 (CH2), 28.30 (CH2), 37.46

(CH2-Ccarborane), 41.38 (CH2N), 41.56 (C-2´), 62.76 (C-5´), 72.11 (C-3´), 75.36 (Ccarborane-

C), 87.16 (C-1´), 88.90 (C-4´), 89.45(Ccarborane-C), 110.70 (C-5), 136.51 (C-6), 152.29 (C-

+ 2), 165.37 (C-4); MS (HR-ESI) C16H33B10N3O5Na (M+Na) calcd: 478.3316, found:

478.3309; HPLC retention time: 20.81 min (Method C), 19.88 min (Method D).

O N NH2 HO N O O

3-[5-(7-Amino-closo-m-carboran-1-yl)pentan-1-yl]thymidine (4.17.A).

Compound 4.17.A was prepared according to the procedure described for compound 4.14.A using compound 4.11 (330 mg, 0.58 mmol) and trifluoroacetic acid

(0.78 mL, 1.16 mmol). Purification by silica gel column chromatography (ethyl

1 acetate/methanol, 15:1) gave 4.17.A as a white solid in 81% (220 mg) yield. Rf 0.20; H-

4 NMR (MeOH-d ) δ 1.22-1.30 (m, 2H, CH2), 1.34-1.45 (m, 2H, CH2), 1.51-1.60 (m, 2H,

CH2), 1.89 (d, 3H, CH3, J = 1.2 Hz), 1.92-1.99 (m, 2H, CH2-Ccarborane), 2.19-2.25 (m, 2H,

H-2´), 3.71 (dd, 1H, H-5´ J = 12.0, 3.7 Hz), 3.79 (dd, 1H, H-5´ J = 12.0, 3.7 Hz), 3.85-

163 3.93 (m, 3H, H-4´ and CH2N), 4.36-4.41 (m, 1H, H-3´), 6.29 (t, 1H, H-1´, J = 6.5 Hz),

13 6 7.82 (d, 1H, H-6, J = 1.2 Hz); C-NMR (acetone-d ) δ 13.20 (CH3), 27.37 (CH2), 28.04

(CH2), 30.73 (CH2), 37.79 (CH2-Ccarborane), 41.31 (CH2N), 42.01 (C-2´), 62.75 (C-5´),

72.09 (C-3´), 75.50 (Ccarborane-C), 87.11 (C-1´), 88.86 (C-4´), 89.41(Ccarborane-C), 110.71

(C-5), 136.47 (C-6), 152.30 (C-2), 165.41 (C-4); MS (HR-ESI) C17H35B10N3O5Na

(M+Na)+ calcd: 492.3472, found: 492.3459; HPLC retention time: 22.65 min (Method

C), 21.25 min (Method D).

O NH2 N HO N O O

3-[5-(7-Amino-closo-m-carboran-1-yl)hexan-1-yl]thymidine (4.18.A).

Compound 4.18.A was prepared according to the procedure described for compound 4.14.A using compound 4.12 (84 mg, 0.14 mmol) and trifluoroacetic acid

(0.19 mL, 0.28 mmol). Purification by silica gel column chromatography (ethyl

1 acetate/methanol, 15:1) gave 4.18.A as a white solid in 78% (53 mg) yield. Rf 0.21; H-

4 NMR (MeOH-d ) δ 1.21-1.28 (m, 4H, CH2), 1.33-1.39 (m, 2H, CH2), 1.53-1.58 (m, 2H,

CH2), 1.89 (d, 3H, CH3, J = 1.1 Hz), 1.91-1.96 (m, 2H, CH2-Ccarborane), 2.15-2.29 (m, 2H,

H-2´), 3.71 (dd, 1H, H-5´, J = 12.1 & 3.4 Hz), 3.79 (dd, 1H, H-5´, J = 12.1, 3.4 Hz), 3.86-

3.91 (m, 3H, CH2N and H-4´), 4.37-4.39 (m, 1H, H-3´), 6.30 (t, 1H, H-1´, J = 6.9 Hz),

13 4 7.83 (d, 1H, H-6, J = 1.1 Hz); C-NMR (MeOH-d ) δ 13.20 (CH3), 27.46 (CH2), 28.32

164 (CH2), 29.77 (CH2), 30.67 (CH2), 31.02 (CH2), 37.90 (CH2-Ccarborane), 41.33 (CH2N),

42.15 (C-2´), 62.76 (C-5´), 72.10 (C-3´), 75.58 (Ccarborane-C), 87.11 (C-1´), 88.87 (C-4´),

89.40 (Ccarborane-C), 110.71 (C-5), 136.46 (C-6), 152.31 (C-2), 165.41 (C-4); MS (HR-

+ ESI) C18H37B10N3O5Na (M+Na) calcd: 506.3629, found: 506.3811; HPLC retention

time: 24.76 min (Method C), 23.35 min (Method D).

O O N NH2 HO N O O

3-[2-(7-Amino-closo-m-carboran-1-yl)ethoxyethyl]thymidine (4.19.A).

Compound 4.19.A was prepared according to the procedure described for compound 4.14.A using compound 4.13 (60 mg, 0.10 mmol) and trifluoroacetic acid

(0.14 mL, 0.20 mmol). Purification by silica gel column chromatography (ethyl

1 acetate/methanol, 15:1) gave 4.19.A as a white solid in 85% (40 mg) yield. Rf 0.13; H-

4 NMR (MeOH-d ) δ 1.91 (d, 3H, CH3, J = 1.2 Hz), 2.07-2.17 (m, 2H, CH2-Ccarborane), 2.21-

2.29 (m, 2H, H-2´), 3.37 (m, 2H, OCH2, J = 5.7 Hz), 3.59 (t, 2H, OCH2, J = 5.6 Hz), 3.71

(dd, 1H, H-5´, J = 12.0, 3.7 Hz), 3.79 (dd, 1H, H-5´, J = 12.0, 3.7 Hz), 3.88-3.92 (m, 2H,

H-4´), 4.14 (m, 2H, CH2N, J = 5.7 Hz), 4.36-4.41 (m, 1H, H-3´), 6.32 (t, 1H, H-1´, J =

13 4 6.9 Hz), 7.85 (d, 1H, H-6, J = 1.2 Hz); C-NMR (MeOH-d ) δ 13.30 (CH3), 37.73 (CH2-

Ccarborane), 41.37 (CH2N), 42.43 (C-2´), 62.78 (C-5´), 67.90 (CH2), 70.08 (CH2), 72.15 (C-

3´), 73.26 (Ccarborane-C), 87.16 (C-1´), 88.89 (C-4´), 89.74 (Ccarborane-C), 110.77 (C-5),

165 + 136.70 (C-6), 152.44 (C-2), 165.53 (C-4); MS (HR-ESI) C16H33B10N3O6Na (M+Na) calcd: 494.3264, found: 494.3250; HPLC retention time: 18.08 min (Method C), 17.61 min (Method D).

O H NH3 N HO N O O

3-[2-(7-Ammonium-nido-m-carboran-1-yl)ethan-1-yl]thymidine(4.14.B).

Tetrabutylbutylammonium fluoride hydrate (200 mg, 0.63 mmol) was added to a

solution of compound 4.14.A (50 mg, 0.12 mmol) in THF (2 mL) and stirred at 70 °C for

1 h. The progress of the reaction was monitored by the IR. Distilled water (5 mL) was

added at 0 °C, followed by the addition of 3N-hydrochloric acid to adjust the pH to 2-3.

The solution was extracted with ethyl acetate (20 mL × 5) and the combined organic

layers were washed with brine, and dried over magnesium sulfate. After filtration and

evaporation, the residue was purified by silica gel column chromatography (ethyl acetate/

methanol, 15:1, 1% acetic acid) to give compound 4.14.B in 70% (35 mg) yield. Rf 0.10;

1 4 H-NMR (MeOH-d ) δ -1.94 (br s, 1H, Hµ), 1.90 (d, 3H, CH3, J = 1.1 Hz), 1.92-1.97 (m,

2H, CH2-Ccarborane), 2.16-2.32 (m, 2H, H-2´), 3.72 (m, 2H, H-5´), 3.88-3.93 (m, 1H, H-4´),

4.02-4.22 (m, 2H, CH2N), 4.36-4.41 (m, 1H, H-3´), 6.27 & 6.36 (t, 1H, H-1´, J = 6.7 Hz),

13 4 7.81 & 7.82 (d, 1H, H-6, J = 1.1 Hz); C-NMR (MeOH-d ) δ 13.28 (CH3), 36.56 &

36.64 (CH2-Ccarborane), 41.21 & 41.42 (CH2N), 44.70 & 44.82 (C-2´), 62.74 & 92.91 (C-

166 5´), 72.00 & 72.12 (C-3´), 86.94 & 87.28 (C-1´), 88.84 (C-4´), 111.72 & 110.20 (C-5),

136.23 & 136.41 (C-6), 152.32 (C-2), 165.63 & 165.70 (C-4); 11B NMR (MeOH-d4): δ -

+ 33.41, -32.35, -20.19, -15.71, -2.24, -0.58; MS (HR-ESI) C14H30B9N3O5Na (M+Na) calcd: 441.2968, found: 441.2964; HPLC retention time: 14.21 & 14.43 min (Method C),

14.49 & 14.65 min (Method D).

O H N NH3 HO N O O

3-[3-(7-Ammonium-nido-m-carboran-1-yl)propan-1-yl]thymidine (4.15.B).

Compound 4.15.B was prepared according to the procedure described for compound 4.14.B using compound 4.14.A (25 mg, 0.057 mmol) and tetrabutylbutylammonium fluoride hydrate (77 mg, 0.33 mmol). Purification by silica gel column chromatography (ethyl acetate/methanol, 15:1, 1% acetic acid) gave 4.15.B in

1 4 61% (15 mg) yield. Rf 0.12; H-NMR (MeOH-d ) δ -1.86 (br s, 1H, Hµ) 1.54-1.82 (m,

4H, CH2-Ccarborane and CH2), 1.90 (d, 3H, CH3, J =1.1 Hz), 2.14-2.30 (m, 2H, H-2´), 3.72

(dd, 1H, H-5´, J =12.0, 3.1 Hz), 3.80 (dd, 1H, H-5´, J =12.0, 3.1 Hz), 3.89-3.94 (m, 3H,

H-4´ and CH2N), 4.36-4.42 (m, 1H, H-3´), 6.30 (t, 1H, H-1´, J = 6.8 Hz), 7.82 (d, 1H, H-

13 4 6, J = 1.1 Hz); C-NMR (MeOH-d ) δ 13.23 (CH3), 31.74 (CH2), 37.01 (CH2-Ccarborane),

41.35 (CH2N), 42.81 (C-2´), 62.76 (C-5´), 72.07 (C-3´), 87.11 (C-1´), 88.85 (C-4´),

111.75 (C-5), 136.38 (C-6), 152.32 (C-2), 165.45 (C-4); 11B-NMR (MeOH-d4): δ -36.85,

167 + -35.15, -23.02, -18.93, -5.02, -3.16; MS (HR-ESI) C15H32B9N3O5Na (M+Na) calcd:

455.3125, found: 455.3123; HPLC retention time: 14.96 min (Method C), 14.91 min

(Method D).

O H NH3 N HO N O O

3-[4-(7-Ammonium-nido-m-carboran-1-yl)butan-1-yl]thymidine (4.16.B).

Compound 4.16.B was prepared according to the procedure described for

compound 4.14.B using compound 4.16.A (45 mg, 0.099 mmol) and

tetrabutylbutylammonium fluoride hydrate (105 mg, 0.33 mmol). Purification by silica

gel column chromatography (ethyl acetate/methanol, 15:1, 1% acetic acid) gave 4.16.B in

1 4 66% (29 mg) yield. Rf 0.13; H-NMR (MeOH-d ) δ -1.92 (br s, 1H, Hµ) 1.64-1.75 (m,

6H, CH2-Ccarborane and CH2), 1.90 (s, 3H, CH3), 2.05-2.33 (m, 2H, H-2´), 3.72 (dd, 1H, H-

5´, J = 12.0, 3.7 Hz), 3.79 (dd, 1H, H-5´, J = 12.0, 3.7 Hz), 3.88-3.91 (m, 3H, H-4´ and

13 CH2N), 4.36-4.41 (m, 1H, H-3´), 6.29 (t, 1H, H-1´, J = 6.6 Hz), 7.82 (s, 1H, H-6); C-

4 NMR (MeOH-d ) δ 13.24 (CH3), 28.81 (CH2), 31.18 (CH2), 39.57 (CH2-Ccarborane), 41.38

(CH2N), 42.58 (C-2´), 62.76 (C-5´), 72.08 (C-3´), 87.15 (C-1´), 88.87 (C-4´), 110.76 (C-

5), 136.41 (C-6), 152.34 (C-2), 165.48 (C-4); 11B-NMR (MeOH-d4) δ -33.94, -32.18, -

+ 20.29, -16.10, -1.88, -0.52; MS (HR-ESI) C16H34B9N3O5Na (M+Na) calcd: 469.3282,

found: 469.3274; HPLC retention time: 15.38 min (Method C), 15.43 min (Method D).

168

O H N NH3 HO N O O

3-[5-(7-Ammonium-nido-m-carboran-1-yl)pentan-1-yl]thymidine (4.17.B).

Compound 4.17.B was prepared according to the procedure described for

compound 4.14.B using compound 4.17.A (50 mg, 0.11 mmol) and

tetrabutylbutylammonium fluoride hydrate (125 mg, 0.40 mmol). Purification by silica

gel column chromatography (ethyl acetate/methanol, 15:1, 1% acetic acid) gave 4.17.B in

1 4 65% (33 mg) yield. Rf 0.15; H-NMR (MeOH-d ) δ -1.88 (br s, 1H, Hµ), 1.32-1.38 (m,

2H, CH2), 1.56-1.78 (m, 6H, CH2-Ccarborane and CH2), 1.90 (d, 3H, CH3, J = 1.2 Hz), 2.13-

2.33 (m, 2H, H-2´), 3.72 (dd, 1H, H-5´, J = 12.0, 3.8 Hz), 3.79 (dd, 1H, H-5´, J = 12.0,

3.8 Hz) 3.84-3.86 (m, 3H, CH2N and H-4´), 4.36-4.41 (m, 1H, H-3´), 6.23 (t, 1H, H-1´, J

13 4 = 6.9 Hz), 7.82 (d, 1H, H-6, J = 1.2 Hz); C NMR (MeOH-d ) δ 13.24 (CH3), 28.28

(CH2), 28.69 (CH2), 33.69 (CH2), 39.73 (CH2-Ccarborane), 41.34 (CH2N), 42.55 (C-2´),

62.77 (C-5´), 72.09 (C-3´), 87.15 (C-1´), 88.87 (C-4´), 110.77 (C-5), 136.44 (C-6),

+ 152.34 (C-2), 165.49 (C-4); MS (HR-ESI) C17H36B9N3O5Na (M+Na) calcd: 483.3420,

+ found: 483.3420, C17H35B9N3O5Na2 (M-H+Na2) calcd: 505.3240, found: 505.3240;

HPLC retention time: 16.36 min (Method C), 16.12 min (Method D).

169 O H NH3 N HO N O O

3-[6-(7-Ammonium-nido-m-carboran-1-yl)hexan-1-yl]thymidine (4.18.B).

Compound 4.18.B was prepared according to the procedure described for

compound 4.14.B using compound 4.18.A (40 mg, 0.083 mmol) and

tetrabutylbutylammonium fluoride hydrate (100 mg, 0.32 mmol). Purification by silica

gel column chromatography (ethyl acetate/methanol, 15:1, 1% acetic acid) gave 4.18.B in

1 4 53% (21 mg) yield. Rf 0.17; H-NMR (MeOH-d ) δ -1.89 (br s, 1H, Hµ) 1.30-1.38 (m,

4H, CH2), 1.64-1.79 (m, 6H, CH2-Ccarborane and CH2), 1.90 (d, 3H, CH3, J = 1.1 Hz), 2.13-

2.29 (m, 2H, H-2´), 3.71 (dd, 1H, H-5´, J = 12.0, 3.0 Hz), 3.80 (dd, 1H, H-5´, J = 12.0,

3.0 Hz), 3.90-3.92 (m, 3H, H-4´ and CH2N), 4.36-4.41 (m, 1H, H-3´), 6.29 (t, 1H, H-1´, J

13 4 = 6.5 Hz), 7.82 (d, 1H, H-6, J = 1.1 Hz); C-NMR (MeOH-d ) δ 13.23 (CH3), 28.06

(CH2), 28.63 (CH2), 30.69 (CH2), 33.94 (CH2), 39.78 (CH2-Ccarborane), 41.34 (CH2N),

42.45 (C-2´), 62.77 (C-5´), 72.11 (C-3´), 87.12 (C-1´), 88.88 (C-4´), 110.74 (C-5), 136.43

(C-6), 152.33 (C-2), 165.46 (C-4); 11B-NMR (MeOH-d4) δ -33.87, -32.15, -20.15, -15.94,

+ -1.91, -0.32; MS (HR-ESI) C18H38B9N3O5Na (M+Na) calcd: 497.3597, found: 497.3596;

HPLC retention time: 17.77 min (Method C), 17.23 min (Method D).

170 O H O N NH3 HO N O O

3-[2-(7-Ammonium-nido-m-carboran-1-yl)ethoxyethyl]thymidine (4.19.B).

Compound 4.19.B was prepared according to the procedure described for

compound 4.14.B using compound 4.19.A (60 mg, 0.13 mmol) and

tetrabutylbutylammonium fluoride hydrate (150 mg, 0.48 mmol). Purification by silica

gel column chromatography (ethyl acetate/methanol, 13:1, 1% acetic acid) gave 4.19.B in

1 4 50% (30 mg) yield. Rf 0.11; H-NMR (MeOH-d ) δ -1.94 (br s, 1H, Hµ) 1.55-1.69 (m,

2H, CH2-Ccarborane), 1.90 (d, 3H, CH3, J = 1.1 Hz), 2.15-2.33 (m, 2H, H-2´), 3.54-3.63 (m,

4H, OCH2) 3.70 (dd, 1H, H-5´, J = 12.1, 3.2 Hz), 3.79 (dd, 1H, H-5´, J = 12.1, 3.2 Hz),

3.88-3.92 (m, 1H, H-4´) 4.13 (t, 2H, CH2N, J = 6.2 Hz), 4.36-4.41 (m, 1H, H-3´), 6.29 (t,

1H, H-1´, J = 6.8 Hz), 7.81 (d, 1H, H-6, J = 1.1 Hz); 13C-NMR (MeOH-d4) δ 13.25

(CH3), 38.82 (CH2-Ccarborane), 41.38 (CH2N), 41.40 (C-2´), 62.76 (C-5´), 67.77 (CH2),

72.06 (C-3´), 74.57 (CH2), 87.18 (C-1´), 88.86 (C-4´), 110.68 (C-5), 136.57 (C-6), 152.41

(C-2), 165.52 (C-4); 11B-NMR (MeOH-d4): δ -33.86, -32.26, -20.23, -16.08, -2.00, -0.57;

+ MS (HR-ESI) C16H34B9N3O6Na (M+Na) calcd: 485.3232, found: 485.3232; HPLC

retention time: 14.48 min (Method C), 14.63 min (Method D).

171 H H N H

7-Amino-7, 9-nido-m-carborane (4.21)

Compound 4.21 was prepared from m-carborane by applying the procedure

121 1 reported by Kahl et al. Rf 0.32 (hexanes/ethylacetate, 1:1); H-NMR (CD3CN) δ 3.23

13 (br s, 1H, H-Ccarborane), 3.78 (br s, 2H, NH2); C-NMR (CD3CN) δ 54.65 (H-Ccarborane),

11 -1 90.45 (N-Ccarborane); B NMR (CD3CN) δ -12.14, -11.56, -9.57, -6.99, -0.62; IR (cm )

+ 3392 (m), 3332, 2595 (s), 1611 (m), 1218, 1054; MS (HR-ESI) C2H14B9N1Na1 (M+H) calcd: 160.2125, found: 160.2134.

H H H H N H

7-Ammomium-7, 9-nido-m-carborane (4.22)

To a solution of tetrabutylammonium fluoride hydrate (2.50 g, 7.94 mmol) in

THF (25 mL) was slowly added a solution of compound 4.21 (420 mg, 2.64 mmol) in

THF (5 mL) at 70 °C. The solution was stirred at the same temperature for 1.5 h. After

cooling to room temperature, distilled water (10 mL) was added, followed by evaporation

of excess THF. Aqueous 3N hydrochloric acid (3 mL) was added to adjust the pH to 2-3.

The solution was extracted with ethyl acetate (30 mL × 2) and the combined ethylacetate layers were washed with brine and dried over magnesium sulfate. After filtration and 172 evaporation, the residue was purified by silica gel column chromatography (hexanes/ ethylacetate, 1:1) to give compound 4.22 in 61% (240 mg, 1.61 m mol) yield. Rf 0.24;

1 H-NMR (CD3CN) δ -1.99 (br s, 1H, bridging H), 1.43 (br s, 1H, H-Ccarborane), 6.04 (br s,

13 11 3H, NH3); C-NMR (CD3CN) δ 32.83 (H-Ccarborane), 53.77 (N–Ccarborane); B-NMR

-1 (CD3CN) δ -33.59, -31.95, -20.07, -19.53, -15.75, -3.60, -2.82, 0.82; IR (cm ) 3215 (s),

+ 3189, 2535 (s), 1467 (s); MS (HR-ESI) C2H14B9N1Na1 (M+Na) calcd: 173.1900, found:

173.1897.

H H N H

7-Isopropylideneammonium-7, 9-nido-m-carborane (4.23)

Compound 4.22 (50 mg, 0.34 mmol) was dissolved in anhydrous acetone (5 mL)

and stirred at room temperature for 6 hours. After evaporation of excess acetone, the

residue was purified by silica gel flash column chromatography (hexanes/ethylacetate,

1 1:1) to give compound 4.23 in 95% yield. Rf 0.27; H-NMR (CD3CN) δ -1.92 (br s, 1H,

bridging H), 1.54 (br s, 1H, H-Ccarborane), 2.35 (s, 3H, CH3), 2.51 (s, 3H, CH3), 10.37 (br s,

13 3H, C=NH); C-NMR (CD3CN) δ 23.09 (CH3), 25.56 (CH3), 32.64 (H-Ccarborane), 61.59

11 (N–Ccarborane), 193.08 (C=N); B-NMR (CD3CN) δ -32.93 , -31.59, -19.75, -19.33, -

18.47, -15.59, -2.48, -2.04, 0.21; IR (cm-1) 3268 (s), 2529 (s), 1662 (s), 1424, 1003; MS

+ (HR-ESI) C5H18B9N1Na1 (M+Na) calcd: 213.2215, found: 213.2216.

173 H H N H

7-Cyclohexylideneammonium-7, 9-nido-m-carborane (4.24)

Compound 4.22 (100 mg, 0.68 mmol) and cyclohexanone (0.13 mL, 1.36 mmol)

were dissolved in anhydrous benzene (3 mL) and stirred at room temperature for 6 h. the precipitated solid was collected by filtration and washed with hexanes to give compound

1 4.24 in 68% yield. Rf 0.24 (hexanes/ethylacetate, 2:1); H-NMR (CD3OD) δ -1.92 (br s,

1H, bridging H), 1.43 (br s, 1H, H-Ccarborane), 3.93 (s, 3H, CH3), 4.04 (s, 3H, CH3), 6.67

(d, 1H, ArH, J = 2.2 Hz), 6.75 (dd, 1H, ArH, J = 8.8, 2.2 Hz), 7.67 (d, 1H, ArH, J = 8.8

13 Hz), 8.45 (s, 1H, CH=N), 10.68 (br s, 1H, NH); C-NMR (CD3OD) δ 25.30 (CH2), 28.08

(CH2), 28.72 (CH2), 33.15 (H-Ccarborane), 33.75 (CH2), 35.82 (CH2), 62.57 (N-Ccarborane),

+ 196.08 (C=N); MS (HR-ESI) C8H22B9N (M+Na) calculated: 252.2561, found: 252.2570.

OCH3 H H H N OCH H 3

7-(2,4-Dimethoxy)benzylideneammonium-7, 9-nido-m-carborane (4.25)

Compound 4.22 (100 mg, 0.68 mmol) and 2,4-dimethoxybenzaldehyde (0.2 mL,

1.20 mmol) were dissolved in anhydrous benzene (3 mL) and stirred at room temperature

174 for 6 hours. The precipitated solid was collected by filtration and washed with hexanes to

1 give compound 4.25 in 61% yield. Rf 0.29 (hexanes/ethylacetate, 2:1); H-NMR

(CD3CN) δ -1.92 (br s, 1H, bridging H), 1.57 (br s, 1H, H-Ccarborane), 3.93 (s, 3H, CH3),

4.04 (s, 3H, CH3), 6.67 (d, 1H, ArH, J = 2.2 Hz), 6.75 (dd, 1H, ArH, J = 8.8, 2.2 Hz),

7.67 (d, 1H, ArH, J = 8.8 Hz), 8.45 (s, 1H, CH=N), 10.68 (br s, 1H, NH); 13C-NMR

(CD3CN) δ 33.02 (H-Ccarborane), 57.32 (OCH3), 57.90 (OCH3), 65.10 (N-Ccarborane), 99.74

(ArC), 109.30 (ArC), 109.99 (ArC), 139.86 (ArC), 163.86 (C=N), 164.88 (ArC), 167.37

+ (ArC); MS (HR-ESI) C11H22B9NO2 (M+Na) calculated: 320.2458, found: 320.2447.

7.4. Chapter 5

Compounds 5.1-5.3 were prepared from dThd by adapting procedures reported

previously.287,288 The reported 1H- and 13C-NMR data of 5.1-5.3 were identical with those obtained by us.

O N O S O O O

5´-O-Tosylthymidine (5.1)

1 6 H-NMR (DMSO-d ) δ 1.77 (s, 3H, CH3), 2.06-2.16 (m, 2H, H-2´), 2.41 (s, 3H,

CH3), 3.85-3.88 (m, 1H, H-4´), 4.13-4.17 (m, 1H, H-3´), 4.16 (dd, 1H, H-5´, J = 10.7, 5.5

Hz), 4.25 (dd, 1H, H-5´, J =10.7, 3.2 Hz), 5.42 (d, 1H, OH, J = 4.3 Hz), 6.14 (t, 1H, H-1´,

175 J =7.0 Hz), 7.38 (s, 1H, H-6), 7.47 (d, 2H, ArH, J = 8.3 Hz), 7.79 (d, 2H, ArH, J = 8.3

13 6 Hz), 11.3 (s, 1H, NH); C-NMR (DMSO-d ) δ 12.04 (CH3), 21.07 (CH3), 38.32 (C-2´),

69.90 (C-5´), 70.12 (C-3´), 83.17 (C-1´), 83.96 (C-4´), 109.76 (C-5), 127.57 (ArC),

130.15 (ArC), 132.10 (ArC), 135.84 (C-6), 145.10 (ArC), 150.32 (C-2), 163.60 (C-4).

N O

N3 O

5´-Azido-5´-deoxythymidine (5.2)

1 6 H-NMR (DMSO-d ) δ 1.79 (s, 3H, CH3), 2.04-2.28 (m, 2H, H-2´), 3.50 (d, 2H,

H-5´, J = 5.0 Hz), 3.81-3.87 (m, 1H, H-4´), 4.17-4.21 (m, 1H, H-3´), 5.39 (d, 1H, OH, J

= 4.2 Hz), 6.20 (t, 1H, H-1´, J = 6.9 Hz), 7.49 (s, 1H, H-6), 11.3 (s, 1H, NH); 13C-NMR

(CD3OD) δ 12.05 (CH3), 38.05 (C-2´), 51.67 (C-5´), 70.70 (C-3´), 83.84 (C-1´), 84.51

(C-4´), 109.77 (C-5), 136.03 (C-6), 150.44 (C-2), 163.62 (C-4).

N O

H2N O

176 5´-Amino-5´-deoxythymidine (5.3)

1 6 H-NMR (DMSO-d ) δ 1.79 (s, 3H, CH3), 2.02-2.13 (m, 2H, H-2´), 2.72 (d, 2H,

NH2), 3.50-3.58 (m, 2H, H-5), 3.62-3.65 (m, 1H, H-4´), 4.16-4.21 (m, 1H, H-3´), 5.14 (s,

1H, OH), 6.14 (t, 1H, H-1´, J = 6.9 Hz), 7.65 (s, 1H, H-6); 13C NMR (DMSO-d6) δ 12.05

(CH3), 38.05 (C-2´), 51.66 (C-5´), 70.70 (C-3´), 83.83 (C-1´), 84.51 (C-4´), 109.77 (C-5),

136.03 (C-6), 150.43 (C-2), 163.61 (C-4)

General procedure for the synthesis of sulfonamide-linked BaTK/BaTMPK

inhibitors (5.4-5.7)

To a solution of 5´-amino-5´-deoxythymidine 5.3 (60 mg, 0.25 mmol) and the

indicated sulfonylchloride (0.28 mmol) [see below] in DMF (5 mL) was added

triethylamine (0.5 mL). The resulting solution was stirred for 6 h at room temperature.

The solution was cooled to 4 °C, triethylamine hydrochloride was removed by filtration,

and the filtrate was concentreated. The residue was triturated with methanol to give a

crystalline solid, which was collected by filtration. Washing the solid with

hexanes/ethylacetate (1:1) afforded the following products.

O O N O H S S N O O O

5´-Deoxy-5´-[4-(methylsulfonyl)benzenesulfonamido]thymidine (5.4) 177 Reaction of 4-(methylsulfonyl)benzenesulfonyl chloride with 5.3 provided

1 compound 5.4 (92 mg, 80%). Rf 0.10 (dichloromethane/methanol, 10:1); H-NMR

6 (DMSO-d ) δ 1.78 (s, 3H, CH3), 2.00-2.15 (m, 2H, H-2´), 2.98 (dd, 1H, H-5´, J = 13.6,

6.7 Hz), 3.11 (dd, 1H, H-5´, J = 13.6, 4.1 Hz), 3.29 (s, 3H, SO2CH3), 3.68-3.72 (m, 1H,

H-4´), 4.12-4.15 (m, 1H, H-3´), 5.33 (d, 1H, OH, J = 4.3 Hz), 6.11 (t, 1H, H-1´, J = 6.8

Hz), 7.49 (s, 1H, H-6), 8.04 (d, 2H, ArH, J = 8.4 Hz), 8.13 (d, 2H, ArH, J = 8.4 Hz), 8.21

13 6 (s, 1H, NH), 11.25 (s, 1H, NH); C-NMR (DMSO-d ) δ 12.10 (CH3), 38.19 (C-2´),

43.14 (C-5´), 44.71 (SO2CH3), 70.84 (C-3´), 83.86 (C-1´), 84.69 (C-4´), 109.70 (C-5),

127.48 (ArC), 128.12 (ArC), 136.28 (C-6), 144.00 (ArC), 145.15 (ArC), 150.44 (C-2),

+ 163.72 (C-4); MS (HR-ESI) C17H21N3O8S2 (M+Na) calcd 482.0668, found 482.0672.

O NH S O O N O H S N O O

5´-Deoxy-5´-[3-(methylsulfonyl)benzenesulfonamido]thymidine (5.5)

Reaction of 3-(methylsulfonyl)benzenesulfonyl chloride with 5.3 provided

1 compound 5.5 (89 mg, 78%). Rf 0.16 (dichloromethane/methanol, 10:1); H-NMR

4 (MeOH-d ) δ 1.89 (d, 3H, CH3, J =1.1 Hz), 2.00-2.23 (m, 2H, H-2´), 3.10-3.25 (m, 2H,

H-5´), 3.18 (s, 1H, SO2CH3), 3.78-3.84 (m, 1H, H-4´), 4.27-4.33 (m, 1H, H-3´), 6.14 (t,

1H, H-1´, J = 6.8 Hz), 7.52 (d, 1H, H-6, J = 1.1 Hz), 7.82 (t, 1H, ArH, J = 7.9 Hz), 8.17

(d, 1H, ArH, J = 7.9 Hz), 8.18 (d, 1H, ArH, J = 7.9 Hz), 8.40 (t, 1H, ArH, J = 1.7 Hz); 178 13 4 C-NMR (MeOH-d ) δ 12.38 (CH3), 40.18 (C-2´), 44.17 (C-5´), 45.77 (SO2CH3), 72.47

(C-3´), 86.23 (C-1´), 86.61 (C-4´), 111.88 (C-5), 126.86 (ArC), 131.79 (ArC), 132.22

(ArC), 132.83 (ArC), 138.30 (C-6), 143.46 (ArC), 143.88 (ArC), 152.28 (C-2), 166.39

+ (C-4); MS (HR-ESI) C17H21N3O8S2 (M+Na) calcd 482.0668, found 482.0666.

O NH S O O N O H S N O O

5´-Deoxy-5´-[2-(methylsulfonyl)benzenesulfonamido]thymidine (5.6)

Reaction of 2-(methylsulfonyl)benzenesulfonyl chloride with 5.3 provided

1 compound 5.6 (83 mg, 72%). Rf 0.16 (dichloromethane/methanol, 10:1); H-NMR

4 (MeOH-d ) δ 1.88 (d, 3H, CH3, J = 1.1 Hz), 2.15-2.25 (m, 2H, H-2´), 3.17 (dd, 1H, H-5´,

J = 13.4, 6.1 Hz), 3.24 (dd, 1H, H-5´, J = 13.4, 3.9 Hz), 3.40 (s, 1H, SO2CH3), 3.82-3.86

(m, 1H, H-4´), 4.26-4.30 (m, 1H, H-3´), 6.11 (t, 1H, H-1´, J = 6.9 Hz), 7.42 (d, 1H, H-6, J

= 1.1 Hz), 7.86-7.90 (m, 2H, ArH), 8.21-8.24 (m, 1H, ArH), 8.27-8.31 (m, 1H, ArH);

13 4 C-NMR (MeOH-d ) δ 12.46 (CH3), 40.10 (C-2´), 44.54 (C-5´), 45.90 (SO2CH3), 72.45

(C-3´), 85.85 (C-1´), 86.41 (C-4´), 111.94 (C-5), 132.73 (ArC), 133.82 (ArC), 134.92

(ArC), 135.57 (ArC), 138.03 (C-6), 139.68 (ArC), 140.20 (ArC), 152.17 (C-2), 166.39

+ (C-4); MS (HR-ESI) C17H21N3O8S2 (M+Na) calcd 482.0668, found 482.0673.

179 O

O N O H S N O O

5´-Deoxy-5´-(naphthalene-1-sulfonamido)thymidine (5.7)

Reaction of 1-naphthlenesulfonyl chloride with 5.3 provided compound 5.7 (70

1 4 mg, 65%). Rf 0.15 (dichloromethane/methanol, 10:1); H-NMR (MeOH-d ) δ 1.85 (d, 3H,

CH3, J = 1.1 Hz), 2.12-2.19 (m, 2H, H-2´), 3.08 (dd, 1H, H-5´, J = 14.0, 6.0 Hz), 3.24 (dd,

1H, H-5´, J = 14.0, 4.1 Hz), 3.75-3.81 (m, 1H, H-4´), 4.23-4.27 (m, 1H, H-3´), 6.05 (t, 1H,

H-1´, J = 6.9 Hz), 7.46 (d, 1H, H-6, J =1.1 Hz), 7.53-7.70 (m, 3H, ArH), 8.00 (dd, 1H,

ArH, J = 7.8, 1.5 Hz), 8.12 (d, 1H, ArH, J = 7.8 Hz), 8.20 (dd, 1H, ArH, J = 7.8, 1.5 Hz),

13 4 8.69 (dd, 1H, ArH, J = 7.8, 1.5 Hz); C-NMR (MeOH-d ) δ 12.33 (CH3), 40.08 (C-2´),

45.73 (C-5´), 72.56 (C-3´), 86.30 (C-1´), 86.84 (C-4´), 111.70 (C-5), 125.25 (ArC),

125.87 (ArC), 127.95 (ArC), 129.01 (ArC), 129.47 (ArC), 130.07 (ArC), 130.14 (ArC),

135.19 (ArC), 135.81 (ArC), 136.71 (ArC), 138.45 (C-6), 152.21 (C-2), 166.39 (C-4);

+ MS (HR-ESI) C20H21N3O6S (M+Na) calcd 454.1049, found 454.1031.

General procedure for the synthesis of urea- or thiourea-linked BaTK/BaTMPK

inhibitors (5.8-5.13)

To a solution of 5´-amino-5´-deoxythymidine 5.3 (60 mg, 0.25 mmol) and the

indicatd phenylisocyanate or phenylisothiocyanate (0.28 mmol) [see below] in pyridine

(10 mL) was added 4-(dimethylamino)pyridine (6 mg, 0.05 mmol). The resulting solution 180 was stirred for 12 h at room temperature. The solvent was evaporated and the residue was

purified by silica gel column chromatography using ethylacetate/methanol (25:1) as the

eluent to give the following products.

S N O H H N N O S OH

5´-Deoxy-5´-{[2-(methylthio)anilinothiocarbonyl]amino}thymidine (5.8)

Reaction of 2-(methylthio)phenyl isothiocyanate with 5.3 provided compound 5.8

1 4 (80 mg, 76%). Rf 0.30 (ethylacetate/methanol, 25:1); H-NMR (MeOH-d ) δ 1.88 (s, 3H,

CH3), 2.22-2.25 (m, 2H, H-2´), 2.40 (s, 3H, SCH3), 3.83-3.98 (m, 2H, H-5´), 4.02-4.06

(m, 1H, H-4´), 4.34-4.38 (m, 1H, H-3´), 6.19 (t, 1H, H-1´, J = 6.9 Hz), 7.18 (dd, 1H, ArH,

J = 7.8, 7.4 Hz ), 7.23 (dd, 1H, ArH, J = 7.8, 7.4 Hz), 7.34 (d, 2H, ArH, J = 7.8 Hz ), 7.52

13 4 (s, 1H, H-6); C-NMR (MeOH-d ) δ 12.47 (CH3), 15.42 (CH3), 39.96 (C-2´), 49.65 (C-

5´), 73.09 (C-3´), 86.34 (C-1´), 87.00 (C-4´), 111.81 (C-5), 126.74 (ArC), 128.07 (ArC),

129.09 (ArC), 129.45 (ArC), 126.25 (ArC), 138.12 (C-6), 141.03 (ArC), 152.28 (C-2),

+ 166.43 (C-4), 182.27 (C=S); MS (HR-ESI) C18H22N4O4S2Na (M+Na) calcd 445.0980,

found 445.0995.

181 O

N O H H S N N O S OH

5´-Deoxy-5´-{[3-(methylthio)anilinothiocarbonyl]amino}thymidine (5.9)

Reaction of 3-(methylthio)phenyl isothiocyanate with 5.3 provided compound 5.9

1 4 (77 mg, 73%). Rf 0.31 (ethylacetate/methanol, 25:1); H-NMR (MeOH-d ) δ 1.86 (d, 3H,

CH3, J = 1.0 Hz), 2.22-2.30 (m, 2H, H-2´), 2.45 (s, 3H, SCH3), 3.77-3.98 (m, 2H, H-5´),

4.04-4.10 (m, 1H, H-4´), 4.32-4.38 (m, 1H, H-3´), 6.21 (t, 1H, H-1´, J = 6.9 Hz), 7.02-

7.04 (m, 1H, ArH), 7.06 (t, 1H, ArH, J = 6.9 Hz) 7.23 (d, 1H, ArH, J = 7.9 Hz), 7.27-7.30

13 4 (m, 1H, ArH), 7.50 (d, 1H, H-6, J = 1.0 Hz); C-NMR (MeOH-d ) δ 12.40 (CH3), 15.43

(CH3), 38.85 (C-2´), 50.31 (C-5´), 73.12 (C-3´), 86.10 (C-1´), 87.11 (C-4´), 111.97 (C-5),

121.61 (ArC), 122.71 (ArC), 124.50 (ArC), 130.51 (ArC), 138.16 (C-6), 140.15 (ArC),

141.43 (ArC), 152.30 (C-2), 166.36 (C-4), 182.66 (C=S); MS (HR-ESI) C18H22N4O4S2Na

(M+Na)+ calcd 445.0980, found 445.0995.

N O H H N N O S S OH

5´-Deoxy-5´-{[4-(methylthio)anilinothiocarbonyl]amino}thymidine (5.10) 182 Reaction of 4-(methylthio)phenyl isothiocyanate with 5.3 provided compound

1 4 5.10 (88 mg, 83%). Rf 0.26 (ethylacetate/methanol, 25:1); H-NMR (MeOH-d ) δ 1.87 (s,

3H, CH3), 2.22-2.28 (m, 2H, H-2´), 2.45 (s, 3H, SCH3), 3.82-3.96 (m, 2H, H-5´), 4.04-

4.07 (m, 1H, H-4´), 4.34-4.38 (m, 1H, H-3´), 6.20 (t, 1H, H-1´, J = 6.9 Hz), 7.22-7.27 (m,

13 4 4H, ArH), 7.52 (s, 1H, H-6); C-NMR (MeOH-d ) δ 12.41 (CH3), 16.00 (CH3), 39.84

(C-2´), 49.71 (C-5´), 73.08 (C-3´), 86.17 (C-1´), 87.08 (C-4´), 111.87 (C-5), 126.34

(ArC), 128.43 (ArC), 138.17 (C-6), 152.29 (C-2), 166.38 (C-4), 182.80 (C=S); MS (HR-

+ ESI) C18H22N4O4S2Na (M+Na) calcd 445.0980, found 445.1007.

N O H H S N N O O OH

5´-Deoxy-5´-{[3-(methylthio)anilinocarbonyl]amino}thymidine (5.11)

Reaction of 3-(methylthio)phenyl isocyanate with 5.3 provided compound 5.11

1 6 (69 mg, 68%). Rf 0.36 (ethylacetate/methanol, 15:1); H-NMR (DMSO-d ) δ 1.77 (s, 3H,

CH3), 2.04-2.19 (m, 2H, H-2´), 2.42 (s, 3H, SCH3), 3.19-3.48 (m, 2H, H-5´), 3.76-3.79

(m, 1H, H-4´), 4.15-4.16 (m, 1H, H-3´), 5.33 (d, 1H, OH, J = 4.2 Hz), 6.18 (t, 1H, H-1´, J

= 7.4 Hz), 6.32 (t, 1H, NH, J = 5.8 Hz), 6.78 (d, 1H, ArH, J = 7.8 Hz), 7.05 (d, 1H, ArH,

J = 7.8 Hz), 7.15 (t, 1H, ArH, J = 7.8 Hz) 7.43 (s, 1H, ArH), 7.52 (s, 1H, H-6), 8.58 (s,

13 6 1H, NH), 11.32 (s, 1H, NH); C-NMR (DMSO-d ) δ 12.06 (CH3), 14.57 (CH3), 38.39 183 (C-2´), 41.43 (C-5´), 71.13 (C-3´), 83.74 (C-1´), 85.30 (C-4´), 109.77 (C-5), 114.18

(ArC), 114.60 (ArC), 118.46 (ArC), 129.14 (ArC), 136.07 (C-6), 138.38 (ArC), 140.91

+ (ArC), 150.48 (C-2), 155.12 (C=O), 163.70 (C-4); MS (HR-ESI) C18H22N4O5S (M+Na) calcd 429.1209, found 429.1214

N O H H N N O O S OH

5´-Deoxy-5´-{[4-(methylthio)anilinocarbonyl]amino}thymidine (5.12)

Reaction of 4-(methylthio)phenyl isocyanate with 5.3 provided compound 5.12

1 6 (74 mg, 73%). Rf 0.34 (ethylacetate/methanol, 15:1); H-NMR (DMSO-d ) δ 1.77 (s, 3H,

CH3), 2.04-2.17 (m, 2H, H-2´), 2.40 (s, 3H, SCH3), 3.17-3.43 (m, 2H, H-5´), 3.75-3.78

(m, 1H, H-4´), 4.17-4.21 (m, 1H, H-3´), 5.37 (d, 1H, OH, J = 4.2 Hz), 6.15 (t, 1H, H-1´, J

= 7.4 Hz), 6.88 (t, 1H, NH, J = 5.8 Hz), 7.14 (d, 2H, ArH, J = 8.6 Hz), 7.37 (d, 2H, ArH,

J = 8.6 Hz), 7.66 (s, 1H, H-6), 9.38 (s, 1H, NH), 11.28 (s, 1H, NH); 13C-NMR (DMSO-

6 d ) δ 11.90 (CH3), 16.26 (CH3), 38.50 (C-2´), 40.50 (C-5´), 70.82 (C-3´), 83.39 (C-1´),

85.21 (C-4´), 109.80 (C-5), 118.06 (ArC), 127.96 (ArC), 128.47 (ArC), 136.13 (C-6),

138.60 (ArC), 150.45 (C-2), 155.49 (C=O), 163.71 (C-4); MS (HR-ESI) C18H22N4O5S

(M+Na)+ calcd 429.1209, found 429.1212.

184 O

O N O H H N N O O O OH

5´-Deoxy-5´-{[3-(methoxycarbonyl)anilinocarbonyl]amino}thymidine (5.13)

Reaction of methyl 3-isocyanatobenzoate with 5.3 provided compound 5.13 (54

1 6 mg, 52%). Rf 0.34 (ethylacetate/methanol, 15:1); H-NMR (DMSO-d ) δ 1.77 (s, 3H,

CH3), 2.07-2.16 (m, 2H, H-2´), 3.23-3.46 (m, 2H, H-5´), 3.75-3.80 (m, 1H, H-4´), 3.83 (s,

3H, OCH3), 4.16-4.18 (m, 1H, H-3´), 5.32 (d, 1H, OH, J = 4.2 Hz), 6.18 (t, 1H, H-1´, J =

7.4 Hz), 6.36 (t, 1H, NH, J = 5.8 Hz), 7.36 (t, 1H, ArH, J = 7.9 Hz), 7.49 (dt, 1H, ArH, J

= 7.9, 1.8 Hz), 7.56 (dt, 1H, ArH, J = 7.9, 1.8 Hz), 7.52 (s, 1H, H-6), 8.12 (t, 1H, ArH, J

13 6 = 1.8 Hz), 8.81 (s, 1H, NH), 11.29 (s, 1H, NH); C-NMR (DMSO-d ) δ 11.98 (CH3),

38.17 (C-2´), 40.79 (C-5´), 52.04 (OCH3), 71.11 (C-3´), 83.76 (C-1´), 85.25 (C-4´),

109.74 (C-5), 116.65 (ArC), 118.00 (ArC), 122.07 (ArC), 129.02 (ArC), 130.04 (ArC),

136.02 (C-6), 140.79 (ArC), 150.45 (C-2), 155.09 (C=O), 163.69 (C-4), 166.25 (C=O);

+ MS (HR-ESI) C19H22N4O7 (M+Na) calcd 441.1386, found 441.1391.

General procedure for the synthesis of amide-linked BaTK/BaTMPK inhibitors

(5.14-5.19)

To a solution of 5´-amino-5´-deoxythymidine 5.3 (60 mg, 0.25 mmol) and the

indicated benzoic acid (0.30 mmol) [See below] in DMF (8 mL) was added 1-

hydroxybenzotriazole (HOBt, 41 mg, 0.30 mmol). A solution of 1,3- 185 dicyclohexylcarbodiimide (DCC, 62 mg, 0.30 mmol) in DMF (2 mL) was added slowly

and the resulting reaction mixture was stirred for 12 h at room temperature. The solvent

was removed under reduced pressure and the residue was purified by silica gel column

chromatography using ethylacetate/methanol (20:1) as the eluent to give the following

products.

O NH H N 2 S N O O H N O O OH

5´-Deoxy-5´-[4-(sulfamoyl)benzeneamido]thymidine (5.14)

Reaction of 4-(sulfamoyl)benzoic acid with 5.3 provided compound 5.14 (51 mg,

1 6 48%). Rf 0.22 (ethylacetate/methanol, 15:1); H-NMR (DMSO-d ) δ 1.77 (s, 3H, CH3),

2.07-2.18 (m, 2H, H-2´), 3.52-3.55 (m, 2H, H-5´), 3.88-3.90 (m, 1H, H-4´), 4.24-4.26 (m,

1H, H-3´), 5.33 (d, 1H, OH, J = 4.4 Hz), 6.15 (t, 1H, H-1´, J = 6.7 Hz), 7.48 (s, 2H, NH2),

7.52 (s, 1H, H-6), 7.90 (d, 2H, ArH, J = 8.3 Hz), 8.01 (d, 2H, ArH, J = 8.3 Hz), 8.82 (t,

13 6 1H, NH, J = 5.6 Hz), 11.30 (s, 1H, NH); C-NMR (DMSO-d ) δ 11.96 (CH3), 38.50 (C-

2´), 41.67 (C-5´), 71.26 (C-3´), 84.03 (C-1´), 84.71 (C-4´), 109.62 (C-5), 125.60 (ArC),

127.88 (ArC), 136.17 (ArC), 137.11 (C-6), 146.30 (ArC), 150.41 (C-2), 163.68 (C-4),

+ 165.47 (C=O); MS (HR-ESI) C17H20N4O7S (M+Na) calcd 447.0950, found 447.0953.

186 O

O O N O H S C N O O

5´-Deoxy-5´-[4-(methylsulfonyl)benzeneamido]thymidine (5.15)

Reaction of 4-(methylsulfonyl)benzoic acid with 5.3 provided compound 5.15 (56

1 6 mg, 53%). Rf 0.27 (ethylacetate/methanol, 15:1); H-NMR (DMSO-d ) δ 1.76 (s, 3H,

CH3), 2.07-2.17 (m, 2H, H-2´), 3.28 (s, 3H, SO2CH3), 3.54-3.58 (m, 2H, H-5´), 3.90-3.94

(m, 1H, H-4´), 4.25-4.29 (m, 1H, H-3´), 5.40 (d, 1H, OH, J = 4.3 Hz), 6.15 (t, 1H, H-1´, J

= 6.8 Hz), 7.53 (s, 1H, H-6), 8.02-8.18 (m, 4H, ArH), 9.02 (s, 1H, NH), 11.38 (s, 1H,

13 6 NH); C-NMR (DMSO-d ) δ 12.10 (CH3), 38.11 (C-2´), 42.14 (C-5´), 44.19 (SO2CH3),

70.89 (C-3´), 83.81 (C-1´), 84.56 (C-4´), 110.11 (C-5), 127.40 (ArC), 128.64 (ArC),

139.50 (C-6), 143.33 (ArC), 150.64 (C-2), 164.02 (C-4), 165.62 (ArC); MS (HR-ESI)

+ C18H21N3O7S (M+Na) calcd 446.0998, found 446.1002.

O N O H S C N O

5´-Deoxy-5´-[4-(methylthio)benzeneamido]thymidine (5.16)

187 Reaction of 4-(methylthio)benzoic acid with 5.3 provided compound 5.16 (57 mg,

1 6 58%). Rf 0.37 (ethylacetate/methanol, 15:1); H-NMR (DMSO-d ) δ 1.76 (s, 3H, CH3),

2.07-2.12 (m, 2H, H-2´), 2.51 (s, 3H, SCH3), 3.49-3.52 (m, 2H, H-5´), 3.88-3.90 (m, 1H,

H-4´), 4.22-4.26 (m, 1H, H-3´), 5.32 (d, 1H, OH, J = 4.3 Hz), 6.14 (t, 1H, H-1´, J = 6.8

Hz), 7.32 (d, 2H, ArH, J = 8.4 Hz), 7.51 (s, 1H, H-6), 7.81 (d, 2H, ArH, J = 8.4 Hz), 8.58

13 6 (t, 1H, NH, J = 5.6 Hz), 11.30 (s, 1H, NH); C-NMR (DMSO-d ) δ 12.45 (CH3), 14.59

(CH3), 41.90 (C-2´), 49.08 (C-5´), 71.76 (C-3´), 84.84 (C-1´), 85.21 (C-4´), 110.47 (C-5),

125.48 (ArC), 128.32 (ArC), 130.47 (ArC), 136.88 (C-6), 143.51 (ArC), 150.99 (C-2),

+ 164.59 (C-4), 167.14 (C=O); MS (HR-ESI) C18H21N3O5S (M+Na) calcd 414.1100,

found 414.1104.

O NH

O N O H N O O OH

5´-Deoxy-5´-[4-(methoxycarbonyl)benzeneamido]thymidine (5.17)

Reaction of mono-methyl terephthalate with 5.3 provided compound 5.17 (45 mg,

1 6 43%). Rf 0.37 (ethylacetate/methanol, 15:1); H-NMR (DMSO-d ) δ 1.76 (s, 3H, CH3),

2.08-2.22 (m, 2H, H-2´), 3.51-3.56 (m, 2H, H-5´), 3.88 (s, 3H, OCH3), 3.88-3.92 (m, 1H,

H-4´), 4.24-4.26 (m, 1H, H-3´), 5.31 (d, 1H, OH, J = 4.4 Hz), 6.14 (t, 1H, H-1´, J = 6.7

Hz), 7.51 (s, 1H, H-6), 7.97 (d, 2H, ArH, J = 7.3 Hz), 8.04 (d, 2H, ArH, J = 7.3 Hz), 8.80

13 4 (t, 1H, NH, J = 5.0 Hz), 11.28 (s, 1H, NH); C-NMR (MeOH-d ) δ 11.93 (CH3), 38.36 188 (C-2´), 41.67 (C-5´), 52.33 (OCH3), 71.28 (C-3´), 84.06 (C-1´), 87.73 (C-4´), 109.60 (C-

5), 127.62 (ArC), 129.11 (ArC), 131.79 (ArC), 136.15 (ArC), 138.34 (C-6), 150.41 (C-2),

163.68 (C-4), 165.65 (C=O), 165.68 (C=O), 169.36 (C=O); MS (HR-ESI) C19H21N3O7

(M+Na)+ calcd 426.1277, found 426.1285.

N O H O N O O O OH

5´-Deoxy-5´-[3-(methoxycarbonyl)benzeneamido]thymidine (5.18)

Reaction of methyl hydrogen isophthalate with 5.3 provided compound 5.18 (38

1 4 mg, 38%). Rf 0.36 (ethylacetate/methanol, 15:1); H-NMR (MeOH-d ) δ 1.82 (s, 3H,

CH3), 2.25-2.30 (m, 2H, H-2´), 3.63-3.78 (m, 2H, H-5´), 3.93 (s, 3H, OCH3), 4.02-4.06

(m, 1H, H-4´), 4.34-4.38 (m, 1H, H-3´), 6.22 (t, 1H, H-1´, J = 7.4 Hz), 7.50 (s, 1H, H-6),

7.59 (t, 1H, ArH, J = 7.8 Hz), 8.09 (dt, 1H, ArH, J = 7.8, 1.2 Hz), 8.17 (dd, 1H, ArH, J =

13 4 7.8, 1.2 Hz), 8.50 (t, 1H, ArH, J = 1.2 Hz); C-NMR (MeOH-d ) δ 12.29 (CH3), 40.11

(C-2´), 42.88 (C-5´), 52.86 (OCH3), 73.06 (C-3´), 86.43 (C-1´), 87.03 (C-4´), 111.75 (C-

5), 129.42 (ArC), 130.02 (ArC), 131.88 (ArC), 132.87 (ArC), 133.45 (ArC), 136.04

(ArC), 138.20 (C-6), 152.32 (C-2), 163.36 (C-4), 167.73 (C=O), 169.36 (C=O); MS (HR-

+ ESI) C19H21N3O7 (M+Na) calcd 426.1277, found 426.1289.

189 O

NH O N O N O O OH

5´-Deoxy-5´-(phthalimido)thymidine (5.19)

Reaction of methyl hydrogen phthalate with 5.3 provided compound 5.19 (26 mg,

1 6 28%). Rf 0.57 (ethylacetate/methanol, 15:1); H-NMR (DMSO-d ) δ 1.82 (s, 3H, CH3),

2.07-2.26 (m, 2H, H-2´), 3.78 (dd, 1H, H-5´, J = 14.2, 7.7 Hz), 3.85 (dd, 1H, H-5´, J =

14.2, 5.6 Hz), 3.99-4.04 (m, 1H, H-4´), 4.22-4.26 (m, 1H, H-3´), 5.37 (d, 1H, OH, J = 4.1

Hz), 6.14 (t, 1H, H-1´, J = 6.8 Hz), 7.56 (s, 1H, H-6), 7.83-7.92 (m, 4H, ArH), 11.25 (s,

13 6 1H, NH); C-NMR (DMSO-d ) δ 12.86 (CH3), 38.84 (C-2´), 47.37 (C-5´), 72.44 (C-3´),

83.19 (C-1´), 84.18 (C-4´), 109.55 (C-5), 123.14 (ArC), 131.43 (ArC), 134.54 (ArC),

136.12(C-6), 150.36 (C-2), 163.65 (C-4), 167.86 (C=O); MS (HR-ESI) C18H17N3O6

(M+Na)+ calcd 394.1015, found 394.1024.

General procedure for the synthesis of triazole-linked BaTK/BaTMPK inhibitors

(5.20-5.23)

Compound 5.2 (65 mg, 0.25 mmol) and the indicated alkyne (0.25 mmol) [see

below] were dissolved in a mixture of water and tert-BuOH (6 mL, 1:1). Freshly prepared

sodium ascorbate solution (0.05 mL, 1 M solution in water) was added, followed by the

addition of freshly prepared copper (II) sulfate pentahydrate (0.01 mL, 0.5 M solution in

water). During vigorously stirring of the reaction mixture at room temperature for 48 h, a 190 precipitate formed. The reaction mixture was diluted with 20 mL of water and cooled to

0 °C. A yellow precipitate was collected by filtration and dried in vacuum to give the

following products.

N O N N N O

5´-Deoxy-5´-[4-phenyl-(1,2,3)triazol-1-yl]thymidine (5.20)

Reaction of ethynylbenzene with 5.2 provided compound 5.20 (14 mg, 15%). Rf

1 6 0.18 (dichloromethane/methanol, 10:1); H-NMR (DMSO-d ) δ 1.68 (s, 3H, CH3), 2.07-

2.21 (m, 2H, H-2´), 3.78 (s, 3H, OCH3), 4.11-4.14 (m, 1H, H-4´), 4.29-4.33 (m, 1H, H-

3´), 4.67 (dd, 1H, H-5´, J = 14.3, 6.8 Hz), 4.76 (dd, 1H, H-5´, J = 14.3, 4.5 Hz), 5.53 (d,

1H, OH, J = 4.4 Hz), 6.19 (t, 1H, H-1´, J = 6.8 Hz), 7.24 (s, 1H, H-6), 7.31-7.44 (m, 3H,

ArH), 7.84 (d, 2H, ArH, J = 7.5 Hz), 8.57 (s, 1H, ArH), 11.31 (s, 1H, NH); 13C-NMR

6 (DMSO-d ) δ 11.96 (CH3), 37.85 (C-2´), 51.09 (C-5´), 71.38 (C-3´), 83.72 (C-1´), 84.65

(C-4´), 115.16 (C-5), 122.19 (ArC-triazole), 125.10 (ArC), 127.87 (ArC), 128.87 (ArC),

130.59 (ArC), 138.20 (C-6), 146.35 (ArC-triazole), 152.32 (C-2), 163.36 (C-4); MS (HR-

+ ESI) C18H19N5O4 (M+Na) calcd 392.1335, found 392.1332.

191 F O

N O N N N O

5´-Deoxy-5´-[4-(4-fluorophenyl)-(1,2,3)triazol-1-yl]thymidine (5.21)

Reaction of 1-ethynyl-4-fluorobenzene with 5.2 provided compound 5.21 (13 mg,

1 6 13%). Rf 0.14 (dichloromethane/methanol, 10:1); H-NMR (DMSO-d ) δ 1.69 (s, 3H,

CH3), 2.11-2.21 (m, 2H, H-2´), 4.09-4.14 (m, 1H, H-4´), 4.28-4.32 (m, 1H, H-3´), 4.67

(dd, 1H, H-5´, J = 14.5, 6.3 Hz), 4.76 (dd, 1H, H-5´, J = 14.5, 4.7 Hz), 5.52 (d, 1H, OH, J

= 4.4 Hz), 6.19 (t, 1H, H-1´, J = 6.8 Hz), 7.23 (s, 1H, H-6), 7.28 (t, 2H, ArH, J = 8.7 Hz),

7.88 (dd, 2H, ArH, J = 8.3, 5.7 Hz), 8.55 (s, 1H, ArH), 11.28 (s, 1H, NH); 13C-NMR

6 (DMSO-d ) δ 12.00 (CH3), 37.88 (C-2´), 51.12 (C-5´), 70.52 (C-3´), 83.72 (C-1´), 83.98

(C-4´), 109.82 (C-5), 115.71 (ArC), 115.93 (ArC), 122.12 (ArC-triazole), 127.11 (ArC),

127.19 (ArC), 135.95 (C-6), 145.51 (ArC-triazole), 150.44 (C-2), 160.55 (ArC), 162.98

+ (ArC), 163.57 (C-4); MS (HR-ESI) C18H18FN5O4 (M+Na) calcd 410.1241, found

410.1241.

O O

N O N N N O

192

5´-Deoxy-5´-[4-(4-methoxyphenyl)-(1,2,3)triazol-1-yl]thymidine (5.22)

Reaction of 4-ethynylanisole with 5.2 provided compound 5.22 (11 mg, 10%). Rf

1 6 0.18 (dichloromethane/methanol, 10:1); H-NMR (DMSO-d ) δ 1.69 (s, 3H, CH3), 2.07-

2.21 (m, 2H, H-2´), 3.78 (s, 3H, OCH3), 4.08-4.12 (m, 1H, H-4´), 4.28-4.32 (m, 1H, H-

3´), 4.65 (dd, 1H, H-5´, J = 14.2, 6.7 Hz), 4.73 (dd, 1H, H-5´, J = 14.2, 4.5 Hz), 5.53 (d,

1H, OH, J = 4.3 Hz), 6.19 (t, 1H, H-1´, J = 6.8 Hz), 7.00 (d, 2H, ArH, J = 8.7 Hz), 7.24 (s,

1H, H-6), 7.76 (d, 2H, ArH, J = 8.7 Hz), 8.45 (s, 1H, ArH), 11.31 (s, 1H, NH); 13C-NMR

6 (DMSO-d ) δ 12.04 (CH3), 38.75 (C-2´), 51.94 (C-5´), 56.00 (OCH3), 71.44 (C-3´), 83.34

(C-1´), 84.65 (C-4´), 109.78 (C-5), 115.16 (ArC), 124.11 (ArC-triazole), 127.36 (ArC),

134.93 (ArC), 135.91 (C-6), 147.18 (ArC-triazole), 152.32 (C-2), 159.88 (ArC), 163.53

+ (C-4); MS (HR-ESI) C19H21N5O5 (M+Na) calcd 422.1440, found 422.1452.

O N NH

N O N N N O

5´-Deoxy-5´-[4-(pyridine-3-yl)-(1,2,3)triazol-1-yl]thymidine (5.23)

Reaction of 3-ethynylpyridine with 5.2 provided compound 5.23 (7 mg, 8%). Rf

1 6 0.06 (dichloromethane/methanol, 8:1); H-NMR (DMSO-d ) δ 1.68 (s, 3H, CH3), 2.08-

2.20 (m, 2H, H-2´), 4.09-4.15 (m, 1H, H-4´), 4.27-4.33 (m, 1H, H-3´), 4.67 (dd, 1H, H-5´,

J = 14.3, 6.5 Hz), 4.79 (dd, 1H, H-5´, J = 14.3, 4.9 Hz), 5.46 (d, 1H, OH, J = 4.3 Hz),

193 6.19 (t, 1H, H-1´, J = 6.8 Hz), 7.24 (s, 1H, H-6), 7.39-7.66 (m, 2H, ArH), 8.23 (d, 1H,

ArH, J = 7.4 Hz), 8.69 (s, 1H, ArH), 8.86-8.89 (m, 1H, ArH), 11.29 (s, 1H, NH); MS

+ (HR-ESI) C17H18N6O4 (M+Na) calcd 393.1287, found 393.1310.

7.5. Phosphoryl transfer assays (PTAs)

Chapter 3 and 4: PTAs with purified recombinant hTK1 and TK2, were carried out as

described previously with minor modifications.59,61 dThd and 3CTAs were dissolved in

DMSO to produce stock solutions of 10 and 100 µM concentrations. The reaction

mixtures contained 10 µM of compounds for hTK1 assays and 100 µM of 3CTA for TK2

assays, 100 µM ATP with 0.03 µM [γ-32P]-ATP (Amersham Pharmacia Biotech, IL, USA),

50 mM Tris-HCl (pH 7.6), 5 mM MgCl2, 125 mM KCl, 10 mM DTT, and 0.5 mg/mL

bovine serum albumin (BSA). The final concentrations of DMSO were set to 1%. The

reaction mixtures were incubated at 37 °C for 20 min in presence of 50 ng of enzyme.

Subsequently, the enzyme was heat-inactivated for 2 min at 95 °C. The reaction mixtures

were centrifuged and 1 µL sample portions were spotted on PEI-cellulose TLC plates

(Merck, USA). The TLC plates were placed overnight in a solvent system (isobutyric acid/ ammonium hydroxide/ water, 66:1:33). The radiolabeled spots were visualized by phospho-imaging (Fuji Film, Science Lab., Image Gauge V3.3) and phosphorylarion values of 3CTAs were expressed relative to that of dThd.

Alternatively, PRs in hTK1 were determined by measuring the change of ADP production in absorbance at 340 nm, caused by NADH oxidation in a coupled enzyme system with pyruvate kinase and lactate dehydrogenase. The standard reaction mixture

contained 40 µM 3CTA (N5-2OH isomers, Chapter 3), 20 mM Tris-HCl (pH 7.6), 50

194 mM KCl, 2 mM MgCl2, 0.5 mM ATP, 5 mM DTT, 1 mM phosphoenolpyruvate, 0.5

units/mL pyruvate kinase, 0.5 units/mL lactate dehydrogenase, 0.1 mM NADH and 0.6

µg enzyme in a total volume of 0.25 mL. The reaction was performed at 24 °C with a

Cary 3 spectrophotometer (Varian Techtron, Mulgrave, Australia) and started by the

addition of dThd or 3CTAs. The enzyme activity values were calculated from the slope

of the absorbance graph. The activity of hTK1 with 20 µM dThd was 640 nmol dTMP

formed per min and mg hTK1 protein.

All measurements were carried out at least three times and the mean and the

standard deviation (SD) were calculated.

Chapter 5: B. anthracis Sterne strain (34F2) tk gene was cloned by Dr. Andrew Phipps

and coworkers at the OSU Department of Veterinary Biosciences. Recombinant BaTK

was expressed and purified by Dr. Staffan Eriksson and coworkers at the Swedish

University of Agricultural Sciences in Uppsala, Sweden. The standard PTAs contained

100 µM nucleoside, 50 mM Tris-HCl (pH 7.6), 2 mM MgCl2, 2 mM ATP, 100 µM ATP

with 0.03 µM [γ-32P]-ATP (Amersham Pharmacia Biotech, IL, USA), 0.5 mg/mL BSA, 5

mM DTT, 15 mM NaF and 10 ng of BaTK in a total volume of 50 µL. The monophosphate products were separated by thin layer chromatography and quantified by phospho-imaging (Fuji Image Gauge V3.3)

Alternatively, BaTK activity with the nucleosides was followed by the ADP production in a coupled enzyme system with pyruvate kinase and lactate dehydrogenase.

The standard reaction mixture contained 50 mM Tris-HCl (pH 7.6), 2 mM MgCl2, 1 mM

ATP, 5 mM DTT, 1 mM phosphoenolpyruvate, 5 units/mL pyruvate kinase, 5 units/mL lactate dehydrogenase, 100 µM NADH and 5 µg BaTK in a total volume of 0.5-1 mL. 195 The reaction was performed at 37 °C with a Cary 3 spectrophotometer (Varian Techtron,

Mulgrave, Australia).

All measurements were carried out at least three times and the mean and the

standard deviation (SD) were calculated.

7.6. In vitro toxicity studies

B. anthracis Sterne was cultured in M9 minimal media with amino acids for 12 to

18 h at 37 ºC with constant shaking. An aliquot of the overnight culture was diluted with fresh M9 minimal media with amino acids to an optical density in the range of 0.050 to

0.100 at 600 nm. Each compound was dissolved in DMSO to produce the corresponding stock solutions of 200 or 500 µM concentrations, respectively. Stock solutions of low concentrations (0.1 µM - 100 µM) were prepared by diluting the initial stock solutions with the M9 minimal media. Each stock solution was freshly prepared before the culture experiment. The stock solution (50 µL) and the culture media (5 mL) were mixed and incubated at 37 ºC with constant shaking. An aliquot of the culture was removed at 4, 6 and 20 h and the optical density at 600 nm was determined. The percent of growth

test(A ) inhibition was determined using the formula %I = 600nm x100 . The minimal control(A600nm )

inhibitory concentration (IC50) was determined by linear regression analysis using

Pharmacokinetic compartment model 107 (inhibitory effect sigmoid Emax model)

implemented in WinNonlin Professional (version 5.0.1, Cary, NC). Enrofloxacin

(Baytril®, Bayer Health Care, Shawnee Mission, Kansas) was included at 100 ng/mL as

a positive control.

196 7.7. In vivo uptake experiments (Chapter 4)

L929 (wt) and L929 TK1 (-) tumors were established in nude mice by implanting

106 cells subcutaneously (s.c.) into the flank of the animals. Two weeks later, the tumors had attained a weight of 0.3 to 1.1 g. Mice were injected intratumorally (i.t.) over a period of 2 min. with quantities N5-2OH, 4.15.B, and BPA equivalent to 50 µg boron solubilized in 15 µL of 70% aqueous DMSO, 10 µL of 24% aqueous DMSO, and 15 µL of PBS, respectively. This was followed by a second injection 2 hours later. In both injections, 0.17 mol equivalents of 5-FdUrd was co-injected to suppress the de novo synthesis of dThd nucleotides. Boron concentrations in tissues were determined DCP-

AES.183 Each point represents the arithmetic mean ± SD of 4 mice.

7.8. Preclinical BNCT experiments (Chapter 3)

BNCT was performed 14 days following intracebrebral stereotactic implantation of F98 glioma cells. Rats were transported to the Nuclear Reactor Laboratory at the

Massachusetts Institute of Technology (MIT) and then randomized on the basis of weight into experimental groups of 7-9 animals each as follows: Group I, 500 µg of 10B-enriched

N5-2OH (3.5.B) in 200 µl of 50% DMSO, administered intracerebrally (i.c.) over a period of 24 hours by CED using ALZET minipumps (model #2001D) at a flow rate of 8

µl/h, followed by irradiation with a collimated beam of thermal neutrons at a reactor power of 4.8 MW-min; Group II received 50% DMSO alone by CED, as per Group I, followed by neutron irradiation; and Group III received 500 µg of 3.5.B in 50% DMSO via CED without neutron irradiation. Group IV received 500 µg of 10B-enriched N5-2OH and processed for boron determinations by DCP-AES as described previously.183 All 197 irradiated rats were anesthetized with a mixture of ketamine and xylazine. BNCT was carried out at the MITR-II reactor in the MIT irradiation facility, which produces a beam of high intensity thermal neutrons without any contaminating fast neutrons. After completion of BNCT, the animals were held at MIT for ~ 3 days to allow induced radioactivity to decay before they were returned to The Ohio State University (Columbus,

OH) for clinical monitoring.

7.9. Molecular modeling

7.9.1. Homology modeling

The initial coordinates of each homology model (hTK1, BaTK, and BaTMPK) were constructed with SWISS-MODEL (Version 36.0003) using the indicated template proteins (1XX6 for hTK1, 1W4R for BaTK and 2CCJ for BaTMPK). The obtained coordinates were then transferred to Sybyl 7.1 and hydrogen atoms were added. The hydrogen atom positions were minimized until an RMS of 0.005 kcal·mol-1Å-1 was reached using the Powell method. The homology models were solvated with water molecules using the Solvent/Solvate option.201 The solvated homology models were minimized until the gradient reached 0.05 kcal·mol-1Å-1 was reached using Tripos Force

Field. For docking studies, the active sites were generated by selecting the same amino acid residues that are located within a radius of 12.0 Å from a ligand in the template crystal structure.

7.9.2. Theoretical calculations using Gaussian 03 program

198 Theoretical calculations for 3.11.A, (R)-epimers of 3.11.D-3.11.E, and 4.21-4.23

using the Gaussian 03 program (Gaussian Inc., Wallingford, CT)194 were carried out by

running on an Itanium 2 Cluster at Ohio Super Computer Center. The structures were

fully optimized at HF/6-31G*, HF/ 6-31G**, B3LYP/6-31G*, B3LYP/6-31G**. All

optimized geometries showed real frequencies. NMR chemical shift calculations of 4.22

were performed with the gauge-including atomic orbitals (GIAO) method using the

11 optimized geometries in acetonitrile (CH3CN) solution. Theoretical B-NMR chemical

shifts were computed versus the chemical shift of diborane (16.6 ppm) and then

1 13 converted to the BF3-Et2O scale. H-NMR and C-NMR chemical shifts were referenced to the tetramethylsilane (TMS) standard.

7.9.3. Calculation of polar surface areas (PSAs) and apolar surface areas (APSAs)

Compounds 3.11.A, (R)-epimers of 3.11.D, and (R)-epimer of 3.11.E were

optimizeded as described above with Gaussian 03 and parameters were transferred to

HyperChemTM 7.51 for windows (Hypercube, Inc., Gainesville, FL). The boron atom

type of the carborane cages in the optimized structures was changed into the C.3 carbon

atom type. For (A)PSA calculations, structure parameters were transferred to the VEGA

ZZ 2.0.4 program (Milano, Italy). A probe radius of 0.5 was used for the calculation of

(A)PSAs.

7.9.4. Docking studies using the FlexX module in Sybyl 7.1

Compounds 3.11.A, (R)-epimers of 3.11.D, and (R)-epimer of 3.11.E optimized using Gaussian 03 as described above were saved in mol2 file format and transferred to

199 Sybyl 7.1 (Tripos Inc, St. Lewis, Missouri). Atom and bond types of the compounds were manually adjusted for docking studies in Sybyl 7.1. Specifically, the atom type of hexavalent boron atoms in the carborane cage was modified to the C.3 atom type because parameters for boron atoms of carborane cages are not available in Sybyl 7.1. For docking studies, the active site was generated by selecting the amino acids that are located within a radius of 12.0 Å from ligands in the crystal structures (1W4R for hTK1 and 1E2F for hTMPK). In case of the homology model, the active site was generated by selecting the same amino acid residues that are located within a radius of 12.0 Å from ligands in the corresponding crystal structures or template protein structures (1W4R for hTK1, 1W4R for BaTK and 2CCJ for BaTMPK). Docking of the compound library into the active site was performed with the FlexX module in Sybyl 7.1.

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APPENDIX A

1H-NMR AND 13C-NMR SPECTRA OF 3.5.A

232

1H-NMR and 2D-COSY spectra of 3.5.A 233

13C-NMR and 13C-DEPT135 spectra of 3.5.A 234

2D-HMQC spectrum of 3.5.A

235