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by Lam Mei Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 127-137). / Abstract also in Chinese. / Acknowledgment --- p.I / Table of content --- p.II / List of figures --- p.VII / List of tables --- p.IX / Abbreviation --- p.X / Abstract --- p.XII / 論文摘要 --- p.XIII / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Acquired immunodeficiency syndrome --- p.1 / Chapter 1.1.1 --- Discovery of AIDS --- p.1 / Chapter 1.1.2 --- Definition and symptoms of AIDS --- p.1 / Chapter 1.1.3 --- AIDS transmission --- p.2 / Chapter 1.1.4 --- AIDS epidemic --- p.3 / Chapter 1.2 --- Human immunodeficiency virus --- p.3 / Chapter 1.2.1 --- Discovery of HIV --- p.3 / Chapter 1.2.2 --- The structure of HIV --- p.4 / Chapter 1.2.3 --- Genomic structure of HIV --- p.5 / Chapter 1.2.4 --- Life cycle of HIV --- p.5 / Chapter 1.2.5 --- How HIV is involved in different stages of AIDS --- p.7 / Chapter 1.3 --- Therapeutic targets for treatment of AIDS --- p.8 / Chapter 1.3.1 --- HIV reverse transcriptase (HIV RT) --- p.8 / Chapter 1.3.2 --- HIV integrase (HIV IN) --- p.11 / Chapter 1.3.3 --- HIV protease (HIV PR) --- p.12 / Chapter 1.3.4 --- Chemokine receptors --- p.14 / Chapter 1.3.5 --- Vaccine development --- p.16 / Chapter 1.4 --- AIDS therapy --- p.17 / Chapter 1.4.1 --- Current status of AIDS therapy --- p.17 / Chapter 1.4.1.1 --- Drugs approved by US Food & Drug Administration (FDA) --- p.17 / Chapter 1.4.1.2 --- Combination therapy --- p.19 / Chapter 1.4.1.3 --- Vaccine development --- p.19 / Chapter 1.4.2 --- Alternative treatment --- p.20 / Chapter 1.5 --- Objective of my project --- p.21 / Chapter Chapter 2 --- Screening of traditional Chinese medicinal (TCM) plants for HIV reverse transcriptase inhibition --- p.22 / Chapter 2.1 --- Introduction --- p.22 / Chapter 2.1.1 --- HIV RT structure and function --- p.22 / Chapter 2.1.2 --- Natural product against HIV RT --- p.25 / Chapter 2.1.3 --- Inhibitory activities from plant extracts --- p.27 / Chapter 2.2 --- Materials and Methods --- p.28 / Chapter 2.2.1 --- Materials --- p.28 / Chapter 2.2.2 --- Extraction methods --- p.30 / Chapter 2.2.2.1 --- Methanol extraction --- p.30 / Chapter 2.2.2.2 --- Hot water extraction --- p.30 / Chapter 2.2.2.3 --- Preparation of Prunella vulgaris extract --- p.30 / Chapter 2.2.3 --- Reverse transcriptase assay --- p.31 / Chapter 2.2.4 --- Characterization of active component in extract of Prunella vulgaris --- p.32 / Chapter 2.2.4.1 --- Protease digestion --- p.32 / Chapter 2.2.4.2 --- Glucosidase digestion --- p.32 / Chapter 2.2.4.3 --- Ethanol precipitation --- p.33 / Chapter 2.2.4.4 --- Sodium periodiate oxidization --- p.33 / Chapter 2.2.4.5 --- Polyvinylpyrrolidone (PVP) Precipitation --- p.34 / Chapter 2.2.4.6 --- Polyamide resin binding --- p.34 / Chapter 2.2.5 --- Purification of Prunella vulgaris extract --- p.34 / Chapter 2.2.5.1 --- Polyamide resin column chromatography --- p.34 / Chapter 2.2.5.2 --- Sephadex LH-20 chromatography --- p.35 / Chapter 2.2.5.3 --- Reverse phase HPLC chromatography --- p.36 / Chapter 2.2.6 --- Characterization of purified Prunella vulgaris extract --- p.37 / Chapter 2.2.6.1 --- Paper chromatography --- p.37 / Chapter 2.2.6.2 --- Acid hydrolysis of extract --- p.37 / Chapter 2.2.6.3 --- Thin layer chromatography --- p.38 / Chapter 2.2.6.4 --- Other assays --- p.39 / Chapter 2.2.7 --- Calculation --- p.40 / Chapter 2.3 --- Results --- p.41 / Chapter 2.3.1 --- Screening of Herbs --- p.41 / Chapter 2.3.1.1 --- Screening of methanol extracts --- p.41 / Chapter 2.3.1.2 --- Screening of hot water extracts --- p.41 / Chapter 2.3.2 --- Characterization of active components in Prunella vulgaris crude extracts --- p.44 / Chapter 2.3.2.1 --- Protease digestion --- p.44 / Chapter 2.3.2.2 --- Glucosidase digestion --- p.44 / Chapter 2.3.2.3 --- Ethanol precipitation --- p.44 / Chapter 2.3.2.4 --- Sodium periodate oxidation --- p.48 / Chapter 2.3.2.5 --- Effect of naturally occurring chemicals on inhibition of HIV RT --- p.48 / Chapter 2.3.2.6 --- Effect of removal of polyphenolic components of aqueous extract on inhibition of HTV RT --- p.51 / Chapter 2.3.3 --- Further purification of active components in aqueous extract of Prunella vulgaris --- p.53 / Chapter 2.3.3.1 --- Absorption chromatography by polyamide resin --- p.53 / Chapter 2.3.3.2 --- The Sephadex LH-20 chromatography --- p.53 / Chapter 2.3.3.3 --- Reverse phase high performance liquid chromatography --- p.56 / Chapter 2.3.3.4 --- Recovery of extract --- p.59 / Chapter 2.3.3.5 --- Inhibition from extract of various steps of purification --- p.59 / Chapter 2.3.4 --- Characterization of purified aqueous extract of Prunella vulgaris --- p.62 / Chapter 2.3.4.1 --- Paper chromatography --- p.62 / Chapter 2.3.4.2 --- Dose response curve --- p.62 / Chapter 2.3.4.3 --- Acid hydrolysis of purified extract --- p.68 / Chapter 2.3.4.4 --- Identification of monosaccharide in purified extract by Thin layer chromatography (TLC) --- p.71 / Chapter 2.3.5 --- Specificity of the purified extract on polymerase inhibition --- p.75 / Chapter 2.3.5.1 --- Inhibition of purified Prunella vulgaris extract on Taq polymerase --- p.75 / Chapter 2.3.5.2 --- Inhibition of purified Prunella vulgaris extract on Superscript II --- p.75 / Chapter 2.4 --- Discussion --- p.79 / Chapter Chapter 3 --- Screening of inhibitory activities from traditional Chinese medicinal (TCM) plants extracts to HIV protease --- p.86 / Chapter 3.1 --- Introduction --- p.86 / Chapter 3.1.1 --- HIV Protease structure and function --- p.86 / Chapter 3.1.2 --- Natural products against HIV Protease --- p.87 / Chapter 3.1.3 --- Plant extracts against HIV Protease --- p.89 / Chapter 3.2 --- Materials and Methods --- p.91 / Chapter 3.2.1 --- Materials --- p.91 / Chapter 3.2.2 --- Expression of HIV protease --- p.92 / Chapter 3.2.2.1 --- Expression and purification of HIV protease --- p.92 / Chapter 3.2.2.2. --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.94 / Chapter 3.2.3 --- Characterization of HIV protease --- p.95 / Chapter 3.2.3.1 --- HIV protease assay by fluorometric measurement --- p.95 / Chapter 3.2.3.2 --- HIV protease assay by using reverse phase high performance liquid chromatography --- p.96 / Chapter 3.3 --- Results --- p.98 / Chapter 3.3.1 --- Expression of HIV protease --- p.98 / Chapter 3.3.2 --- HIV protease assay --- p.98 / Chapter 3.3.2.1 --- Protease assay by using reverse phase HPLC --- p.98 / Chapter 3.3.2.2 --- Protease assay by fluorometric measurement --- p.98 / Chapter 3.3.3 --- Screening of crude Chinese medicinal extracts on inhibition of HIV protease --- p.104 / Chapter 3.3.3.1 --- Methanol extracts --- p.104 / Chapter 3.3.3.2 --- Water extracts --- p.105 / Chapter 3.3.4 --- Characterization of herbal extracts on inhibition of HIV protease --- p.110 / Chapter 3.3.4.1 --- Dose response curve of methanol extract of Woodwardia unigemmata --- p.110 / Chapter 3.3.4.2 --- Dose response curve of hot water extract of Prunella vulgaris --- p.110 / Chapter 3.3.4.3 --- Inhibition mode of methanol extract of Woodwardia unigemmata --- p.113 / Chapter 3.3.4.4 --- Inhibition mode of hot water extract of Prunella vulgaris --- p.113 / Chapter 3.3.4.5 --- Effect of partially purified extracts on HIV protease inhibition --- p.116 / Chapter 3.4 --- Discussion --- p.119 / Chapter Chapter 4 --- General discussion --- p.124 / References --- p.127 / Appendix / Appendix 1 Pictures of herbs used in this study --- p.i / Appendix 2 Mass spectrometry of purified Prunella vulgaris extract --- p.vi / Appendix 3 Calibration curve for determination of HIV PR concentration --- p.viii

Les nucléosides modifiés représentent aujourd'hui une famille incontournable dans la chimiothérapie antivirale. Leur développement progressif au cours de ces 50 dernières années a permis d'endiguer de nombreuses épidémies et d'apporter des traitements efficaces contre de nombreux virus tels que les herpès, les hépatites ou encore le VIH. Toutefois, les infections virales continuent de représenter un problème de santé publique majeur en raison de l'émergence de souches virales résistante aux traitements existants ainsi que l'apparition de nouveaux virus. A ce titre, le développement de nouveaux antiviraux plus actifs, plus sûrs et/ou possédant des modes d'action alternatifs reste plus que jamais d‘actualité. Ce manuscrit, divisé en deux grandes parties, présente d'abord la synthèse métallo-catalysée de nouvelles familles de dérivés nucléosidiques (acycliques et osidiques) puis s'étend ensuite à la préparation de dérivés hétérocycliques à visée anti-VIH. Ainsi, dans une première partie, l'utilisation de réactions de métathèses croisées, catalysées au Ru et activées par les ultrasons, ainsi que l'emploi de lipases dans des réactions de protections/déprotections régiosélectives nous ont permis d'élaborer deux nouvelles familles de nucléosides acycliques alkényles. D'autre part, des réactions d'hétérocouplages acétyléniques catalysées au Ni/Cu ont été réalisés dans le cadre de la synthèse d'une bibliothèque de 2'-déoxyuridines portant un motif 1,3-diyne en position C5. Dans une deuxième partie, la réaction multicomposante de Biginelli a été utilisée dans le développement d'une série de dérivés de dihydropyrimidines β-dicétoacides à visée anti-VIH par inhibition de l'intégrase virale. / Modified nucleosides represent the cornerstone of antiviral chemotherapy. Their progressive development over the last 50 years permitted to contain many epidemics and provided effective treatments against many viruses such as herpes, hepatitis or HIV. However, viral infections remain a major public health problem due to the emergence of resistant strains to existing treatments and the appearance of new viruses. As such, the development of new antivirals, most active and safer and/or acting through alternative mechanisms remains, more than ever, necessary. In this context, the work presented in this manuscript are part of the effort to design and synthesize new molecules with antiviral activities. This manuscript, divided in two parts, firstly focus on the metallo-catalyzed synthesis of new families of nucleoside derivatives (acyclic and osidic) and continue with the synthesis of heterocyclic structures targeting anti-HIV activity. Thus, the use of Ru-catalyzed metathesis reactions under ultrasonic activation and the lipases-catalyzed regioselective protection/deprotection reactions allowed us to develop two new families of alkenyl acyclic nucleosides. The synthesis and antiviral evaluation of C5-(1,3-diyne)-2'-deoxyuridine derivatives, prepared by Ni/Cu-mediated alkyne C-H heterocoupling reaction, are also described. In the second part, the multicomponent Biginelli reaction has been used to develop a series of dihydropyrimidine derivatives bearing a β-diketoacids unit targeting anti-HIV activity by inhibition of the viral integrase.