Chapter 1A Nucleosides, Nucleotides as Antiviral Agents

150 Pages·2006·2.4 MB·English

KATHOLIEKE UNIVERSITEIT LEUVEN
FACULTEIT FARMACEUTISCHE WETENSCHAPPEN

SYNTHESIS AND PROPERTIES OF AMINOPROPYL
NUCLEOSIDES AND NUCLEIC ACIDS

PROEFSCHRIFT TER VERKRIJGING VAN DE GRAAD VAN
DOCTOR IN DE FARMACEUTISCHE WETENSCHAPPEN

door

Ding Zhou

LEUVEN 2006
Monday, September 25, 2006 at 17:00h, Auditorium of the Arenberg Castle,
Kasteelpark Arenberg 1, Heverlee.

Leuven

2006

Promotor: Prof. Piet. Herdewijn
Co-promotor: Prof. Arthur. Van Aerschot

Faculteit Farmaceutische Wetenschappen
Laboratorium voor Medicinale Scheikunde
Rega Instituut
Minderbroedersstraat, 10
B-3000 Leuven
Acknowledgments
This thesis is the result of four years of work whereby I have been accompanied and
supported by many people. It is a pleasant aspect that I have now the opportunity to express
my gratitude for all of them.
The first person I would like to thank is my promoter, Prof. Piet Herdewijn of the Medicinal
Chemistry Laboratory at Rega Institute of KUL. I have been in his group since 2001 when I
started my MSc assignment. During these years I have known Prof. Piet as a sympatic, hard-
working and principle-centered person. His overly enthusiasm, professional and integral view
on research and his mission for providing 'only high-quality work and not less', has made a
deep impression on me. I owe him lots of gratitude for having me shown this way of research.
He could not even realize how much I have learned from him.
I would like to thank my co-promotor Professor Arthur Van Aerschot of the Medicinal
Chemistry Laboratory at Rega Institute of KUL who kept an eye on the progress of my work
and always was available when I needed his advice. Especially the strict and extensive
comments and the many discussions and the interactions with Professor Arthur had a direct
impact on the final form and quality of this thesis. He was even available during the weekend
when he provided valuable comments on the thesis.

I am also very grateful to my co-promotor of my MSc degree, Dr Irene Lagoja, who led me to
go into the door of Medicinal Chemistry. She gave me many discussions, advice and tips that
helped me a lot in staying at the right track.

I also appreciate Prof. Erik De Clercq and Prof. Christophe Pannecouque for examining the
biological activity of compounds in chapter 2, Prof. Roger Busson for checking the NMR,
Prof. Jef Rozenski for making the Mass analysis and Luk Baudemprez for doing 2D NMR
spectrums.
I thank Guy Schepers who synthesized lots of oligonucleotides and did lots of T
m
determinations. I also want to thank Kristof Mullens who did part of T determinations and
m
Luc Kerremans who taught me many pratical operations and gave me support to complete my
research. I also want to thank Chantal Biernaux for excellent editorial help in getting things
formal in a correct way. I am also very grateful to Prof. Helmut Rosemeyer, visiting professor
from University of Osnabrück, Germany, who gave me very important help when I was
looking for a job.
I would like to express my gratitude to all those who gave me support, help, interest and
valuable hints in my research work. Especially I am obliged to Prof. Eveline Lescrinier,
Mikhail Abramov, Mathy Froeyen, Damien Marchand, Martin Hradilek, Alberto Di Salvo,
András Horváth, Fengwu Liu, Bart Ruttens, Mariola Kozlowska, Olga Adelfinskaya, Natalia
Dyubankova, Gert Emmerechts, Pieter Van de Vijver, Peter Buzder-Lantos, Marleen Renders,
Miyeon Jang, Joris Segers, Quiya Huang, Katrijn Bockstael, Catia Lambertucci, Antonietta
Iaconinoto and Sonia Mertens. We not only enjoy the happy time of coffee breaks but also
shared the happiness and sorrow from chemistry together. I thank you all for having shared
many experiences and thoughts with me throughout the last years.
I had the pleasure to work with Tongfei Wu, Lavinia Brennan, Johny Wehbe, Evalina
Colacino, Dolores Viña Castelao, Filip Borgions, Sara Vijgen, Tomasz Ostrowski, Dequn Sun,
Thierry Lioux, Dorothee Bardiot, Ping Gu. Although they already left the lab, their scientific
and friendly help still affect me. I also want to thank my friends in Leuven. They give me nice
memory and happiness all through my stay in Leuven.

I feel a deep sense of gratitude for my parents who formed part of my vision and taught me
the good things that really matter in life.
I am very grateful for my wife Qing Wang, for her love and patience during the PhD period.
One of the best experiences that I lived through in this period was stay with my son Yiquan
Zhou, who provided an additional and joyful dimension to our life mission.
The chain of my gratitude would be definitely incomplete if I would forget to thank the first
cause of this chain, James Zhang, who introduced me to this lab five years ago.
Publications

1. Ding Zhou, I. M. Lagoja, J. Rosenski, R. Busson, A. Van Aerschot and P. Herdewijn.
“Synthesis and Properties of Aminopropyl Nucleic Acids (APNAs)”, Chembiochem, 2005, 6,
2298-2306.

2. Ding Zhou, I. M. Lagoja, A. Van Aerschot and P. Herdewijn. “Synthesis of Aminopropyl
Phosphonate Nucleosides with Purine and Pyrimidine Bases”. Collect. Czech. Chem. Commun.
2006, 71(1), 15-34.

3. Ding Zhou, M. Froeyen, J. Rosenski, A. Van Aerschot and P. Herdewijn. “Chemical
Etiology of Nucleic Acids: Aminopropyl Nucleic Acids”. Accepted by Chemistry &
Biodiversity.
ABBREVIATIONS
ACV acyclovir
AIDS human acquired immunodeficiency syndrome
AMP adenosine monophosphate
ANP acyclic nucleoside phosphonate
ANPpp acyclic nucleoside triphosphate
APN aminopropyl nucleoside
APNA aminopropyl nucleic acids
AZT 3'-azidothymidine
BCH-189 2'-deoxy-3'-thiacytidine
Boc tert-butyloxycarbonyl
Bpoc 2-(biphenyl-4-yl)propan-2-yloxycarbonyl
t-Bumeoc 1-(3,5-di-tert-butylphenyl)-1-methylethoxycarbonyl
BVdU brivudin; 5-bromovinyl-2’-deoxyuridine
BzCl benzoyl chloride
CC cytotoxic concentration
50
CMV cytomegalovirus
CXCR4 chemokine (C-X-C motif) receptor 4
dATP 2’-deoxyadenosine 5’-triphosphate
DCM dichloromethane
dCTP 2’-deoxycytidine 5’-triphosphate
ddC 2',3'-dideoxycytidine
ddI 2',3'-dideoxyinosine
Ddz 2-(3,(-dimethoxyphenyl)propan-2-yloxycarbonyl
DIAD diisopropyl azodicarboxylate
DIPEA diisopropylethylamine
DMDC 2’-deoxy-2’-methylidene-cytidine
DMDFC 2’-deoxy-2’-methylidene-5-fluorocytidine
DMF dimethylformamide
DMSO dimethylsulfoxide
DMTr dimethoxytrityl
DNA deoxyribonucleic acid
dNTPs 2´-deoxynucleoside 5´-triphosphates
d4T 2',3'-didehydro-2',3'-dideoxythymidine
dUTP deoxyuridine triphosphate
EBV Epstein-Barr virus
EdU 5-ethyl –2’-deoxyuridine
ESI electrospray-ionization
Et N triethylamine
3
EtOAc ethyl acetate
3´-Fd4A 3´-fluoro-2',3'-dideoxy-2',3'-didehydroadenosine
FDA food and drug administration
3´-Fd4C 3´-fluoro-2',3'-dideoxy-2',3'-didehydrocytidine
β-L-Fd4C 2',3'-dideoxy-2',3'-didehydro-β-L-5-fluorocytidine
FddClU 5-chloro-2',3'-dideoxy-3'-fluorouridine
FEAU 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-ethyluracil
FeLV feline leukemia virus
FIAU 2’-fluoro-2’-deoxy-β-D-arabinofuranosyl-5-iodouridine
FIV feline immunodeficiency virus
FLT 3'-fluoro-3'-deoxythymidine
FMAU 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-methyluracil
F-OddC 1,3-dioxolane-5-fluorocytidine
FPMP N-[3-fluoro-2-(phosphonomethoxy)propyl]
FTC 5-fluoro- β-L-thiocytidine
GMP guanosine monophosphate
GNA glycol nucleic acid
HBV hepatitis B virus
HCMV human cytomegalovirus
HCV hepatitis C virus
HHV human herpes virus
HIV human immunodeficiency virus
Homo-DNA hexopyranosyl-(4' 6') DNA
HPLC high pressure liquid chromatography
HPMP N-(S)-(3-hydroxy-2-phosphonylmethoxypropyl)
(S)-HPMPA (S)-9-[3-Hydroxy-2-(phosphonomethoxy)propyl]adenine
(S)-HPMPC (S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl]cytosine;
cidofovir
HPMPCpp cidofovir diphosphate
HSV herpes simplex virus
IDU 5-iodo-2’-deoxyuridine
IMP inosine 5'-monophosphate
MesCl mesitylenesulfonyl chloride
MMTrCl (4-methoxyphenyl)diphenylmethyl chloride
MSV moloney murine sarcoma virus
NDP nucleoside diphosphate
NMI 1-methylimidazole
NRTIs nucleoside reverse transcriptase inhibitors
NTPs nucleoside triphosphates
L-OddC β-L-dioxolane cytosine
Ph P triphenylphosphine
3
PK protein kinase
PME N-(2-phosphonylmethoxyethyl)
PMEA 9-[(2-phosphonomethoxy)ethyl]adenine
PMEApp adefovir diphosphate
PMP N-[(2-phosphonomethoxy)propyl
(R)-PMPA (R)-9-[(2-phosphonomethoxy)propyl]adenine; tenofovir
PMPA-DAPy (2-phosphonylmethoxypropyl)oxy-2,4-diaminopyrimidine
PMPApp tenofovir diphosphate
PNAs peptide nucleic acids
RNA ribonucleic acid
p-RNA hexopyranosyl RNA
RT reverse transcriptase
SAH S-adenosylhomocysteine
TBAF tetrabutylammonium fluoride
TBDMSCl tert–butyldimethylsilyl chloride
3TC β-L-thiocytidine
TEAB triethylammonium bicarbonate
TFA triflouroacetic acid
TFT 5-trifluoromethyl-2’deoxyuridine
THF tetrahyfrofuran
4´-thio-F-araA 2´-fluoro-4´-thioarabinofuranosyladenine
4’-thio-F-araDAP 2´-fluoro-4´-thioarabinofuranosyldiaminopurine
4’-thio-F-araG 2´-fluoro-4´-thioarabinofuranosylguanine
TK thymidine kinase
Tm melting temperature, the temperature at which 50 % of a
duplex is dissociated
TMS tetramethylsilane
TMSBr bromotrimethylsilane
TNA (L)-α-threofuranosyl nucleic acids
TPS-Cl 2,4,6-triisopropylbenzenesulfonyl chloride
TsOH toluenesulfonic acid
VV vaccinia virus
VZV varicella zoster virus
CONTENTS
Acknoledgement
Abbreviations
Pubulications
Contents
CHAPTER 1 GENERAL INTRODUCTION......................................................................................1
1.1. NUCLEOSIDES AND NUCLEOTIDES AS ANTIVIRAL AGENTS........................................1
1.1.1. INTRODUCTION...........................................................................................................................1
1.1.2. TARGETS FOR ANTIVIRAL THERAPY............................................................................................2
1.1.3. NUCLEOSIDE ANALOGS AS THERAPEUTIC AGENTS4.....................................................................3
1.1.4. GENERAL MODE OF ACTION OF NUCLEOSIDE ANALOGS..............................................................3
1.1.5. SUGAR-MODIFIED NUCLEOSIDE ANALOGS..................................................................................5
1.1.5.1. 2´-Deoxynucleosides and related drugs.............................................................................5
1.1.5.2. 2’,3’-Dideoxynucleoside analogs and Carbocyclic nucleoside analogs............................6
1.1.5.3. Acyclic nucleoside analogs................................................................................................8
1.1.6. ACYCLIC NUCLEOSIDE PHOSPHONATES....................................................................................10
1.1.6.1. Mechanism of antiviral action.........................................................................................12
1.2. CHEMICAL ETIOLOGY OF THE NUCLEIC ACID STRUCTURE....................................14
1.2.1. INTRODUCTION.........................................................................................................................14
1.2.2. RNA WORLD............................................................................................................................15
1.2.3. CHEMICAL ETIOLOGY OF NUCLEIC ACID STRUCTURE..............................................................17
1.2.3.1. Hexopyranosyl-(4' 6') Oligonucleotide Systems (Homo-DNA).....................................18
1.2.3.2. Pentopyranosyl-(2' 4') Oligonucleotide Systems (p-RNA)............................................20
1.2.3.3. (L)-α-threofuranosyl oligonucleotides (TNA, NH-TNA)..................................................21
1.2.3.4. Peptide nucleic acids (PNAs)...........................................................................................23
1.2.3.5. Acylic flexible backbone- glycol nucleic acid (GNA).......................................................24
1.3. RATIONALE AND OBJECTIVES OF THE STUDY...............................................................25
1.3.1. SYNTHESIS OF AMINOPROPYL PHOSPHONATE NUCLEOSIDES WITH PURINE AND PYRIMIDINE
BASES.................................................................................................................................................25
1.3.2 THE SIMPLEST NUCLEIC ACID ALTERNATIVE: AMINOPROPYL NUCLEIC ACIDS (APNAS)............26
REFERENCES.......................................................................................................................................28
CHAPTER 2 SYNTHESIS OF AMINOPROPYL PHOSPHONATE NUCLEOSIDES WITH
PURINE AND PYRIMIDINE BASES................................................................................................33
2.1. INTRODUCTION............................................................................................................................33
2.2. RESULTS AND DISCUSSION..........................................................................................................34
2.2.1. Synthesis of Aminopropyl Phosphonate Nucleosides 1a-d:................................................34
2.2.2. Synthesis of Aminopropyl Phosphonate Nucleosides 2a-d:................................................36
2.2.3. Synthesis of Aminopropyl Phosphonate Nucleosides 3a-d:................................................37
2.2.4. HPLC purification and analysis of all aminopropyl phosphonate nucleosides 1, 2 and 3.39
2.3. BIOLOGICAL RESULTS..................................................................................................................40
2.4. CONCLUSION...............................................................................................................................41
2.5. EXPERIMENTAL PART..................................................................................................................41
CHAPTER 3 SYNTHESIS AND PROPERTIES OF AMINOPROPYL NUCLEIC ACIDS
(APNA)..................................................................................................................................................60
3.1. INTRODUCTION............................................................................................................................60
3.2. RESULTS AND DISCUSSION..........................................................................................................62
3.2.1. SYNTHESIS OF PROTECTED (R)-ACYCLIC NUCLEOSIDES........................................62
3.2.2. SYNTHESIS OF PROTECTED (S)-ACYCLIC NUCLEOSIDES.........................................64
3.2.3. SYNTHESIS OF THE DIMERS WITH ACYCLIC NUCLEOSIDES....................................65
3.2.3.1. Solution phase synthesis of dimer with phosphoramidite methodology......................................65
3.2.3.2. Solution phase synthesis of dimer with phosphotriester methodology........................................66
3.2.4. SYNTHESIS OF DIMERS FOR OLIGONUCLEOTIDE SYNTHESIS................................67
3.2.5. SYNTHESIS OF OLIGONUCLEOTIDES WITH AYCLIC 3’NH-PHOSPHORAMIDATE
LINKAGE......................................................................................................................................68
3.2.6 PAIRING PROPERTIES OF OLIGONUCLEOTIDE SEQUENCES CONTAINING A 3’-
NH- PHOSPHORAMIDATE LINKAGE.......................................................................................69
3.3. CONCLUSION...............................................................................................................................72
3.4. ADDENDUM: NUCLEOSIDE ANALOGS...........................................................................................73
3.4.1. Synthesis of new acyclic universal nucleoside analogs with 5-nitroindazole.....................73
3.4.2. Synthesis of oligonucleotides with new nucleoside analogs................................................74
3.4.3 Pairing properties of oligonucleotide sequences containing universal nucleosides............75
3.4.4. Conclusion..........................................................................................................................76
3.5. EXPERIMENTAL PART..................................................................................................................77
REFERENCES.......................................................................................................................................96
CHAPTER 4 CHEMICAL ETIOLOGY OF NUCLEIC ACID STRUCTURE: AMINOPROPYL
NUCLEIC ACIDS (APNAS)...............................................................................................................97
4.1. INTRODUCTION............................................................................................................................97
4.2. RESULTS AND DISCUSSION...........................................................................................................99
4.2.1. Synthesis of amino protecting group t-Bumeoc (1-(3,5-di-tert-butylphenyl)-1-
methylethoxycarbonyl) for the building blocks...........................................................................100
4.2.2. Synthesis of protected (R)- and (S)-3’-acyclic nucleosides (3’-APNs)..............................102
4.2.3. Synthesis of protected (R)- and (S)-2’-aminopropyl nucleosides. (2’-APNs)....................103
4.2.4. Synthesis of oligonucleotides with ayclic 3’ or 2’NH -phosphoramidate linkages...........105
4.2.4.1. Synthesis of oligonucleotides with incorporation of 3’-aminopropyl thymidine 19b – Test
reactions.................................................................................................................................................106
4.2.4.2. Synthesis of fully modified oligonucleotides with acyclic 3’ or 2’NH -phosphoramidate linkages
...............................................................................................................................................................109
4.2.5. Pairing properties of the acyclic phosphoramidates.........................................................114
4.3. CONCLUSION.............................................................................................................................116
4.4. EXPERIMENTAL PART................................................................................................................117
REFERENCES.....................................................................................................................................132
GENERAL CONCLUSIONS AND SUMMARY............................................................................133
PART 1: SYNTHESIS OF AMINOPROPYL PHOSPHONATE NUCLEOSIDES WITH PURINE AND PYRIMIDINE
BASES...............................................................................................................................................133
PART 2: SYNTHESIS AND PROPERTIES OF AMINOPROPYL NUCLEIC ACIDS (APNA)..........................134
REFERENCE......................................................................................................................................136
SAMENVATTING.............................................................................................................................137
DEEL 1 : SYNTHESE VAN AMINOPROPYL FOSFONAAT NUCLEOSIDE ANALOGEN MET EEN PURINE OF
PYRIMIDINE BASE.............................................................................................................................137
DEEL 2 : SYNTHESE EN EIGENSCHAPPEN VAN AMINOPROPYL NUCLEÏNEZUREN (APNA)................138
REFERENTIE......................................................................................................................................140