Design, Chemical Synthesis and Biological Evaluation of Potential New Antiviral Agents

190 Pages·2012·7.34 MB·English

by  Andrea Defant

International Doctoral School in Biomolecular Sciences
XXV Cycle

Design, Chemical Synthesis and Biological
Evaluation of Potential New Antiviral Agents

Tutor
Prof. Ines MANCINI
University of Trento, Italy

Ph.D. Thesis of
Andrea DEFANT
University of Trento, Italy

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to my parents

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Contents

List of Abbreviations v
List of Figures ix
List of Schemes xii
List of Tables xv
Abstract 1
1. INTRODUCTION 3
1.1. Structure of HIV virion 4
1.2. HIV life cycle 6
1.3. Possible targets for anti-HIV agents and
clinically used therapeutic agents 7
1.4. Reverse transcriptase enzyme 17
1.5. HIV-1 Non-Nucleoside Reverse Transcriptase
Inhibitors (NNRTIs) 19
1.6. Drug design of new potential NNRTI molecules 25

2. MATERIAL AND METHODS 37
2.1. Computational approach 37
2.2. Chemistry 37
2.2.1 General 38
2.2.2 Instruments 38
2.2.3 Chemical procedures of synthesis
2.3. Biological assays 39
2.3.1. ELISA enzymatic assay 39
2.3.2. In vitro anti-HIV activity 39
2.3.3. In vitro antiviral activity 39
2.3.4. Antibacterial activity 40

3. RESULTS 43
3.1. Drug design 43
3.1.1. Docking calculation 45
3.1.1.1. Proteins preparation 45
3.1.1.2. Preparation of ligands 46
3.1.1.3. Molecular docking 47
3.1.1.4 ADME and drug-like properties prediction 50

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3.2. Chemical synthesis and structural characterization
of the new molecules 55
3.2.1. Precursors 29, 30 and 35 58
3.2.2. Tosylate precursors 59
3.2.2.1. General procedure of synthesis 59
3.2.2.2. Spectroscopic and mass spectrometric data 60
3.2.3. New amino-pyrones 1-6 and 22-23 61
3.2.3.1. General procedure of synthesis 61
3.2.3.2. Spectroscopic and mass spectrometric data 62
3.2.4. New dichloroketo-amino compounds 66
3.2.4.1. General procedure of synthesis 66
3.2.4.2. Spectroscopic and mass spectrometric data 67
3.2.5. New amides 8-10 69
3.2.5.1. General procedure of synthesis 70
3.2.5.2. Spectroscopic and mass spectrometric data 70
3.2.6. New thioethers 14, 16, 25 and 32 71
3.2.6.1. General procedure of synthesis 71
3.2.6.2. Spectroscopic and mass spectrometric data 72
3.2.7. New sulfones 15, 17, 21 and 26 74
3.2.7.1. General procedure of synthesis 74
3.2.7.2. Spectroscopic and mass spectrometric data 74
3.2.8. New ethers 18-20 and 23 76
3.2.8.1. General procedure of synthesis 76
3.2.8.2. Spectroscopic and mass spectrometric data 77
3.2.9. Synthesis and structural characterization of
4-bromo-6-methyl-2H-pyran-2-one (37) 80
3.2.10. Study of the selectivity in N, C-acylation of
4-benzylamino-pyrone 80
3.2.10.1. Synthesis and structural characterization of
compounds 38-43 81
3.2.10.2. Spectroscopic and mass spectrometric data 82
3.2.10.3. Study of selectivity in enamino-ester 45
as a model compound 85
3.2.11. Isolation and purification of drug nevirapine (NVP)
from Viramune® tablets 86
3.3. Biological evaluation 89
3.3.1. ELISA enzymatic assay 89
3.3.1.1. Validation of ELISA assay 90
3.3.1.2. Results of the assay 92
3.3.2. Anti-HIV activity assay 92
3.3.3. Antiviral activity 94
3.3.4. Cytostatic activity 102
3.3.5. Antibacterial activity 103

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4. DISCUSSION 107
4.1. Drug design 107
4.1.1. Docking validation 109
4.1.2. Docking evaluation 111
4.1.3. ADME and drug-like properties prediction 112
4.2. Chemical synthesis and structural characterization of
the new molecules 112
4.3. Study of the selectivity in N,C-acylation of
4-benzylamino-pyrone 120
4.3.1. Regioselectivity in a model enamino-ester 127
4.4. Biological evaluation 128
4.4.1. ELISA enzymatic assay 128
4.4.2. Anti-HIV activity 129
4.4.3. Antiviral activity 131
4.4.4. Cytostatic activity of some molecules 131
4.4.5. Antibacterial activity of some molecules 133

5. CONCLUSION 135

6. REFERENCES 137

Acknowledgments 147

Appendix A: Table I. Results from docking calculations
on a theoretical library of molecules I

Appendix B: Presentation of preliminary results at XIII
International Workshop on Complex Systems VIII

Appendix C: Contribution at the 16th International Electronic X
Conference on Synthetic Organic Chemistry

Appendix D: Additional research activity XII
Appendix E: Curriculum vitae XVII

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List of Abbreviations

ABTS 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid
ADME Absorption, Distribution, Metabolism, Excretion
AIDS Acquired Immuno Deficiency Syndrome
α-APA 2-((2-acetyl-5-methylphenyl)amino)-2-(2,6-
dichlorophenyl)acetamide
ATCC American Type Culture Collection
AZT 3’-azido-2’,3’-didesoxytimidine
CAN cerium(IV) ammonium nitrate
CC Cytotoxic Concentration required to reduce cell growth by
50
50%.
CCID Virus Dose to Infect 50% of the Cell Cultures
50
CCR5 C-C Chemokine Receptor type 5
CDC Centers for Disease Control and Prevention
CrFK Crandell-Rees Feline Kidney
CXCR4 C-X-C chemokine receptor type 4
DABCO 1,4-diazabicyclo[2.2.2]octane
DBU 1,8-diazabicyclo[5.4.0]undec-7-ene
DFT Density Functional Theory
DIG-POD digoxigenin-peroxidase
DIPEA N,N-diisopropylethylamine
DMAP 4-dimethylaminopyridine
DMSO dimethyl sulfoxide
dNTP desoxyribonucleotides triphosphate
DS-5000 dextran sulfate with molecular weight 5000
EC Half Maximal Effective Concentration
50
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EI-MS Electron Impact Mass Spectrometry
ELISA Enzyme-Linked ImmunoSorbant Assay
ESI-MS ElectroSpray Ionization Mass Spectrometry
FC Flash Chromatography
FIPV Feline Corona Virus
FPR False Positive Rate
GA Genetic Algorithm
HAART Highly Active Anti-Retroviral Therapy
HBY (S)-4-isopropoxycarbonyl-6-methoxy-3-methylthiomethyl-3,4-
dihydroquinoxalin-2(1H)-thione
HHA carbohydrate-binding Hippeastrum hybrid
HIA Human Intestinal Absorption
HIV Human immunodeficiency Virus
HMBC Heteronuclear Multiple Quantum Correlation
HR(EI)MS High Resolution Electron Impact ionization Mass Spectrometry
HSQC Heteronuclear Single Quantum Correlation
HSV Herpes Simplex Virus
IC 50 % Inhibitory Concentration (µmol L–1) required to inhibit
50
tumor cell proliferation by 50 %.
Log P Partition Coefficient
LS Local Search
MDCK Madin Darby Canine Kidney
MIC Minimum Inhibitory Concentration
MTP MicroTitre Plate
MRSA Methicillin-Resistant Staphylococcus aureus
MRX 5-bromo-3-(pyrrolidin-1-ylsulfonyl)-1H-indole- 2-carboxamide
NCI National Cancer Institute
NMR Nuclear Magnetic Resonance
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