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Synthesis and Biological Evaluation of Novel Guanine-containing
Compounds as Potential HIV-1 Non-nucleoside Reverse Transcriptase
or Integrase Inhibitors
KASSIM
FOLORUNSHO
ADEBAMBO
Submitted for the Degree of Doctor of Philosophy
Heriot – Watt University
School of Engineering and Physical Sciences
March 2010
The copyright in this thesis is owned by the author. Any quotation from the thesis or use
of any of the information contained in it must acknowledge this thesis as the source of the
quotation or information.
ABSTRACT
The global epidemic of HIV/AIDS has become one of the most pressing public health
emergencies of this century. Various chemotherapy approaches towards the treatment of
HIV-1 have been developed. These approaches were based on targets identified in the
life-cycle of HIV-1. In this research work the synthesis of novel guanine compounds is
described which were tested for their activities towards reverse transcriptase and
integrase enzymes. Initial attempts to synthesise Boc and Cbz protected guan-9-ylacetic
acids from commercially available 2-amino-6-chloropurine were inspired by the work of
Dey and Garner. Their approach was extended to the synthesis N2-mono-Boc-guan-9ylacetic acid in 55% yield.
Furthermore, a novel route was designed to the latter
compound involving the activation of the exocyclic amino group of the purine ring with
triphosgene, followed by addition of tert-butanol.
product in 55% yield.
This approach gave the desired
Although the yields of the two strategies were similar, the time
taken for completion of the second route was much shorter (3 days cf 7 days). An
attempt to synthesize N2-diBoc-guan-9-ylacetic acid only afforded this compound in 20%
yield. The activation of the exocyclic amino group of 2-amino-6-chloropurine with
triphosgene was also explored for the synthesis of N2-Cbz protected guan-9-ylacetic acid.
This gave the desired Cbz-protected N2-guanyl-9-ylacetic acid in yields ranging from
50% to 90% depending on whether ethylbromoacetate or tert-butylbromoacetate was
used. The synthesis of orthogonally protected guanine-PNA monomers is described.
Boc- and Fmoc-protected aminoethylglycine (PNA backbones) were efficiently prepared
using established procedures.
The various intermediates were screened for their
biological activities and it was found out that N2-benzyloxycarbonylguan-9-ylacetic acid
derivatives inhibited HIV-1 reverse transcriptase by interacting with the non-nucleoside
binding pocket and they showed a reduced loss of potency towards common drug
resistant
HIV-1
mutant
strains.
Various
structural
modifications
of
N2-
benzyloxycarbonylguan-9-ylacetic acid derivatives were also prepared. Finally, attempts
were made to synthesize guanine-PNA dimers and guanine-rich PNA oligomers
containing guanine and thymine bases.
ii
DEDICATION
This work is dedicated to God and to over 40 million people living with HIV worldwide.
iii
ACKNOWLEDEMENT
I seize this opportunity to thank the following people for their support during my study,
Professor Brian Wherret and Professor Alan Welch for their moral and financial support
(James Watt Fee Only Scholarship), Professor Dave Adam for his various useful
scientific advices.
The untiring support of my supervisor Dr. Nicola Howarth morally, academically and
socially, gave me the necessary energy needed to complete my PhD work without any
problem. I am very grateful for her honesty, hospitality, and willingness to help at
anytime. I acknowledge the support of the technical staff of the department of Chemistry
Heriot Watt University, namely: Dr. Alan Boyd, for NMR Services, Christina Graham,
for elemental analysis, Dr. Georgina Rosair for X-ray analysis and EU-Frame-work
Project 6 on Targeting Replication and Integration of HIV (TRiOH) for bench and
conference travelling expenses. I am also grateful to my research co-workers in the
Department of Chemistry (Research Associates and PhD students), for their support and
love during the PhD programme.
I acknowledged the contribution of Pastor Christiana Longe of Redeemed Christian
Church of God (Open Heavens Edinburgh) and other ministers of God, for their financial
and spiritual support during my PhD programme.
Finally, I appreciate the support of my family and in-laws during this program. To my
little princesses Boluwatife, Mofeoluwa, Oyinoluwabukunmi and my darling wife
Victoria I say a very big thank you for your support and understanding during the
laboratory work and my write-up period, thank you and God bless you all.
iv
ACADEMIC REGISTRY
Research Thesis Submission
Name:
ADEBAMBO, KASSIM FOLORUNSHO
School/PGI:
ENGINEERING AND PHYSICAL SCIENCES
Version:
FINAL
(i.e. First,
Resubmission, Final)
Degree Sought
(Award and
Subject area)
PHD ORGANIC CHEMISTRY
Declaration
In accordance with the appropriate regulations I hereby submit my thesis and I declare that:
1)
2)
3)
4)
5)
*
the thesis embodies the results of my own work and has been composed by myself
where appropriate, I have made acknowledgement of the work of others and have made
reference to work carried out in collaboration with other persons
the thesis is the correct version of the thesis for submission and is the same version as any
electronic versions submitted*.
my thesis for the award referred to, deposited in the Heriot-Watt University Library, should
be made available for loan or photocopying and be available via the Institutional
Repository, subject to such conditions as the Librarian may require
I understand that as a student of the University I am required to abide by the Regulations of
the University and to conform to its discipline.
Please note that it is the responsibility of the candidate to ensure that the correct version of
the thesis is submitted.
Signature of
Candidate:
Date:
19TH AUGUST 2010
Submission
Submitted By (name in capitals):
ADEBAMBO, KASSIM FOLORUNSHO
Signature of Individual Submitting:
Date Submitted:
19TH AUGUST 2010
For Completion in Academic Registry
Received in the Academic
Registry by (name in capitals):
v
Method of Submission
(Handed in to Academic Registry; posted
through internal/external mail):
E-thesis Submitted (mandatory for
final theses from January 2009)
Signature:
Date:
vi
TABLE OF CONTENTS
Pages
Title page
i
Abstract
ii
Dedication
iii
Acknowledgement
iv
Declaration
v
Table of contents
vi
List of tables and figures
x
List of abbreviations
xiii
List of publications
xv
CHAPTER ONE: INTRODUCTION
1.0
Introduction
1
1.1
Global Epidemic of Human Immunodeficiency Syndrome
3
1.2
Viruses
6
1.3
Classification of Human Immunodeficiency Virus type 1 Strains
8
1.3.1
Origin of HIV-1
9
1.3.2
HIV-1 Life Cycle
11
1.4
Common Chemotherapy of Human Immunodeficiency Virus
Type 1 Infection
21
1.4.1
Viral Entry Inhibitors
22
1.4.2
Reverse Transcriptase Inhibitors
24
1.4.2.1 Nucleoside Reverse Transcriptase Inhibitors
24
1.4.2.2 Non-nucleoside Reverse Transcriptase Inhibitors
26
1.4.3
Resistance of HIV-1 Reverse Transcriptase Enzymes
vii
1.5.
1.6
1.7
to Reverse Transcriptase Inhibitors
29
1.4.4
Highly Active Anti-Retroviral Therapy
29
1.4.5
Integrase Inhibitor Compounds
30
1.4.5.1 The Diketo Aryl Compounds
30
1.4.5.2 Catechol Derivatives
36
1.4.5.3 Other Integrase Inhibitors
38
1.4.6
39
Protease Inhibitors
Guanine-Derived Compounds as Novel HIV-1 Therapeutics
41
1.5.1
Guanine-Derived Compounds as RT inhibitors
41
1.5.2
Guanine-Derived Compounds as IN inhibitors
46
Peptide Nucleic Acids
48
1.6.1
Properties of Peptide Nucleic Acids
50
1.6.2
PNAs as a Potential Anti-HIV-1 Therapeutics
53
Aims and Objectives of the Research
54
CHAPTER TWO: RESULTS AND DISCUSSION
(CHEMICAL SYNTHESIS OF GUANINE CONTAINING COMPOUNDS)
2.1
2.2
Established Routes for the Synthesis of PNA Monomers
58
2.1.1
Traditional Routes to all PNA Monomers
58
2.1.2
Guan-9-ylacetic Acid Synthetic Routes
60
An Attempt to Synthesise N2-Carbamate Protected Guan-9-ylacetic Acids 71
2.2.1
Alternative Routes Investigated towards the Synthesis
of Boc-Protected N9-Guanylacetic Acid
74
2.2.2
Synthesis of N2-Boc Guan-9-ylacetic Acid
76
2.2.3
Optimization of the Route to the Synthesis of Compound 95
78
2.3
Alternative Route Independent of Dey and Garner’s Approach
78
2.4
Synthesis of N2-Benzyloxycarbonyl Guanylacetic Acid
82
2.4.1
Synthesis of N2-Cbz Protected Guanylacetic Acid
via the Isocyanate Intermediate
2.4.2
Effect of Ring Deactivation on the Rate of Reaction of
viii
84
Benzyl Alcohol with an Isocyanate
2.4.3
89
Effect of Increasing in Lipophilicity of the Ester at the
N9- Position on the Purification and Yield of
the desired N2-Benzyloxycarbonyl Protected
Guan-9-ylacetic Acid
2.5
2.6
90
Synthesis of PNA Backbone
93
2.5.1
Synthesis of Fmoc-PNA Backbone
93
2.5.2
Synthesis of Boc-PNA Backbone
95
Synthesis of Guanine PNA Monomers
2.6.1
97
Synthesis of Guanine PNA Monomers for
Fmoc Solid Phase or Solution Phase Strategy
2.6.2
97
Synthesis of Guanine PNA Monomer for
Boc Solid Phase or Solution Phase Strategy
98
2.7
Solution Phase Synthesis of Guanine PNA Dimer
100
2.8
Synthesis of Thymine PNA Monomer
104
2.9
Solid Phase Synthesis of Guanine-rich PNA Oligomers:
2.10
H-TGGG-Lysine-NH2
106
Conclusion
108
CHAPTER
THREE:
EXPLORATION
OF
GUANINE-CONTAINING
COMPOUNDS FOR ANTI-REVERSE TRANSCRIPTASE AND
ANTI-INTEGRASE ACTIVITIES
3.1
HIV-1 Therapeutic Studies of the Purine Compounds
109
3.1.2
Non-nucleoside Reverse Transcriptase Inhibitors
110
3.1.3
Integrase Inhibitors
113
3.2
Evaluation of Guanyl Compounds as Potential NNRTIs
113
3.3
Evaluation of Guanyl Compounds as Potential IN Inhibitors
118
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CHAPTER FOUR: STRUCTURAL ACTIVITY STUDIES OF
THE BIOACTIVE COMPOUNDS
CHAPTER FIVE:
119
CONCLUSION AND FUTURE WORK
5.1
Conclusion
128
5.2
Recommendation
131
CHAPTER SIX:
6.1
6.2
EXPERIMENTAL
Materials and Methods
6.1.1
Reagents and Solvents
132
6.1.2
Chromatography
132
6.1.3
Instrumentation
133
Experimental Procedures
133
Appendix: X-ray data of Compound 103
190
References
201
x
LIST OF TABLES AND FIGURES
LIST OF TABLES
Pages
Table 1:
Global HIV/AIDS Estimates, end of 2006
3
Table 2:
HIV-1 Infections Reported in UK per annum
5
Table 3:
Guanine Compounds that Exhibit NNRTIs Properties
114
Table 4:
Kinetic Parameters for N2-Cbz Guanylacetic Acid
Derivatives Binding to HIV-1 RT Wild Type
115
Table 5:
Anti-viral Assays of the Guanine Scaffolds
118
Table 6:
Crystal Data and Structure Refinement for x80265prev
190
Table 7:
Table 8:
Table 9:
Bond Lengths [Å] and Angles [°] for x80265prev
Torsion Angles [°] for x80265prev
Hydrogen Bonds for x80265prev [Å and °]
191
197
200
LIST OF FIGURES
Pages
Figure 1:
Global Trend of HIV-1
4
Figure 2:
General Structure of Viruses
6
Figure 3:
Diagram Illustrating the Different Levels of HIV Classifications
8
Figure 4:
Structure of HIV-1
11
Figure 5:
HIV Life Cycle
11
Figure 6:
HIV-1 Entry Mechanisms
12
Figure 7:
HIV-1 Reverse Transcriptase Structure
13
Figure 8:
Three Domains of HIV-1 IN
15
Figure 9:
Schematic Illustration of IN-Mediated 3'- End Processing
of Viral DNA and Nucleophilic Attack on host DNA
Figure 10:
17
The Nomenclature P1…Pn, P1’…Pn’ Designate Amino
Acids Residues of Peptide Substrates. The Corresponding Binding Sites
xi
on the Protease are referred to as S1…..Sn; S1’……Sn’
18
Figure 11:
Proposed Catalytic Mechanism for Aspartic Protease
19
Figure 12:
Proposed Concerted Catalytic Mechanism for
HIV-1 Protease Enzyme
20
Figure 13:
HIV-1 Viral Entry Inhibitors
22
Figure 14:
The Amino Acid Sequence of Fuzeon
23
Figure 15:
Inhibition Process of Fuzeon
23
Figure 16:
Structure of Some Current NRTIs
24
Figure 17:
Structures of Some NNRTIs
27
Figure 18:
Structures of Some Second Generation NNRTIs
28
Figure 19:
Structure of a DKA
31
Figure 20:
Chemical Structure of Antiviral Integrase Inhibitors
32
Figure 21
Possible Binding Mode of a Selected Integrase Inhibitor
33
Figure 22:
Proposed Mechanism of Action of DKAs
34
Figure 23:
Structure of Raltegravir
36
Figure 24:
Structures of Some Catechol Derivatives
37
Figure 25:
Structures of Other IN Inhibitors
38
Figure 26:
Structures of Other Potential IN Inhibitors
38
Figure 27:
FDA Approved Protease Inhibitors
40
Figure 28:
Other FDA Approved Protease Inhibitors
41
Figure 29:
Structure of Carbovir
42
Figure 30:
Structure of Analogues of Carbovir
43
Figure 31:
Structure of Cyclobutyl G
43
Figure 32:
Structure of Guanine Prodrugs
44
Figure 33:
Structure of Acyclovir
45
Figure 34:
Structures of Other Antivaral Purine Compounds
46
Figure 35:
Four G-Bases Associated through Hoogsteen
Hydrogen Bonding to Form a Cyclic Structure
Figure 36:
Figure 37:
48
Assembly of Four Guanine Nucleobases into a
G-Tetrad Stabilized by a Central Potassium Ion
48
Structure of Peptide Nucleic Acid
49
xii
Figure 38:
Structure of DNA and RNA
50
Figure 39:
Binding of PNA to DNA using both Watson-Crick and
Hoogsteen Base Pairing in 2:1 Stoichiometry
52
Figure 40:
Complexes of PNA with dsDNA
53
Figure 41:
Guanine PNA Monomers
56
Figure 42:
X-ray Crystallographic Structure of 103
84
Figure 43:
Guanine PNA Dimers
102
Figure 44:
O6-Nitrophenoxy Guanine PNA Dimer
104
Figure 45:
Guanine Compounds Submitted for Bioassay
109
Figure 46:
NNRTIs which are Structurally Similar to our Guanine Scaffolds 112
Figure 47:
Guanine Scaffolds that Exhibits NNRTIs Properties
114
Figure 48:
Enzyme- Ligand Binding Association
114
Figure 49:
Effect of Dilution on the Inhibition of HIV-1 RT by Compound
116
Figure 50:
Resistance Profiles for Compounds 124, 108, Nevirapine
and Efavirenz
117
xiii
LIST OF ABBREVIATIONS
ACN =
Acetonitrile
AZT
=
Azidothymidine
Boc
=
tert-Butoxycarbonyl
Boc2O =
Di-tert-butyl dicarbonate
BOP
Benzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluoro
=
phosphate
Cbz
=
Benzyloxycarbonyl
CCD =
Catalytic core domain
d4T
2’-3’-didehydro-3’-deoxythymidine
=
DABCO =
1,4-Diazabicyclo[2,2,2]octane
DBU =
1,8-Diazabicyclo[5.4.0]undec-7-ene
DCC =
1,3-Dicyclohexylcarbodimide
DCM =
Dichloromethane
ddC =
Dideoxycytosine
DIPEA =
N,N-Diisopropylethylamine
DKAs =
Diketo Acid
DMAP =
4-Dimethylaminopyridine
DMF =
N,N-Dimethylaminoformaldehyde
DNA =
Deoxyribonucleic acid
EC50 =
50% Effective concentration
FDA =
Food Drug Administration
HAART =
Highly Active Anti-Retroviral Therapy
HBTU =
O-Benzotriazol-1yl-N,N,N’,N’-tetramethyluroniumhexafluoro
phosphate
HOBT =
1-Hydroxybenzotriazole hydrate
IC50 =
50% Inhibitory concentration
IN
=
Integrase
MBHA =
4-Methylbenzhydrylamine hydrochloride
Mmt
Monomethoxytrityl
=
xiv
NMR =
Nuclear Magnetic Resonance
NNBS =
Non-nucleoside binding site
NNRTI =
Non-nucleoside Reverse Transcriptase Inhibitors
NRTI =
Nucleoside Reverse Transcriptase Inhibitors
PI
Protease Inhibitors
=
PMEA =
9-[-2(phosphonomethoxy)-ethyl]adenine
PMEG =
9-[-2(phosphonomethoxy)-ethyl]guanine
PNA =
Peptide Nucleic Acids
r.t=
Room Temperature(RT)
RNA =
Ribonucleic acids
RT =
Reverse Transcriptase
TFA =
Trifluoroacetic acid
TFMSA=
Trifluoromethanesulphonic acid
THF
Tetrahydrofuran
=
TLC =
UK
Thin Layer Chromatography
=
UNAIDS =
United Kingdom
Joint United Nations Programme on HIV/AIDS
xv
LIST OF PUBLICATIONS
1.
Adebambo, K.F, Howarth, N.M and Rosair, G.M (2005): Benzyl 2-amino-6chloro-9H-purine-9-carboxylate. Acta Crystallographica, E61, 0486-0488.
2.
Adebambo, K.F; Zanal, S; Thomas, M; Cancio, R; Howarth, N.M; and Maga, G
(2007): N2-Benzyloxycarbonylguan-9-ylacetic acid derivatives inhibit HIV-1
reverse transcriptase by interacting with the non-nucleoside inhibitors binding
pocket and show reduced loss of potency by common drug resistance.
ChemMedChem 2 (10), 1405-1409
xvi