Mitoxantrone is a dihydroxyanthracenedione derivative with significant clinical activity against advanced breast cancer, lymphoma and several types of leukaemia. However, as with all conventional anticancer agents, its non-specific nature results in dose-limiting systemic toxicity. The pharmacological (and toxicological) effects of mitoxantrone are thought to be mediated through its interaction with DNA and the DNA processing enzyme, topoisomerase II. Hence, an investigation of the DNA binding properties of mitoxantrone and related bis-substituted alkylaminoanthraquinones was undertaken with a view to developing agents with reduced toxicity to normal cells. DNA intercalation was evaluated using spectrophotometric and DNA thermal denaturation (Tm) techniques with calf thymus DNA. Overall, compounds that were hydroxylated at the 5 and 8 positions of the anthraquinone chromophore had increased DNA binding affinity compared to their non-hydroxylated analogues. The ~ Tms of mitoxantrone and its non-hydroxylated analogue ametantrone, were 26.4 °C and 21.5°C respectively. The affinity constants (K) for the chromophore-hydroxylated compounds were between 3.94 and 4.95 xl06M-1 while K values for their non-hydroxylated analogues were between 1.63 and 3.25 xl06M-l. For the di-N-oxide, AQ4N, intercalation was not detectable. The mono-N-oxide, AQ6N, showed modest DNA binding activity, with a ~Tm of7.0°C and an affinity constant of 3.64 Xl06M-l. Several of the alkylaminoanthraquinones were further investigated for their ability to inhibit decatenation of kDNA by topoisomerase II. Mitoxantrone and its analogues, AQ4 and AQ6, inhibited decatenation at concentrations ofO.75IlM, 1.51lM and 1.0llM respectively. In contrast, total inhibition of decatenation by the N-oxides, AQ4N and AQ6N, required concentrations of 50llM and 1 OIlM respectively. Hence modification of the terminal nitrogen on both alkylamino side chains to form a di-N-oxide resulted in a large decrease in DNA binding affinity and topoisomerase II inhibition. In view of the importance of the cationic alkyl amino side chains in intercalative binding and the enhanced ability of mitoxantrone to inhibit topoisomerase II, a series of acetalanthraquinones were synthesized. These compounds, referred to as YCG7 (1- substituted), YCG8 (l,4-bis-substituted), and YCG9 (l,5-bis-substituted), possessed dimethoxy groups in place of the alcohol groups of mitoxantrone, and were designed to be converted to their respective aldehydes, which should increase their cytotoxic activity due to their potential to form Schiffs bases with intracellular targets. The acetalanthraquinones were relatively poor DNA intercalators compared to the parent compound, ametantrone. The ~Tm values for YCG7, YCG8 and YCG9 were 3.6 °C, 14.2 °C and 15.7 °C respectively. The bathochromic shifts for the acetalanthraquinones in the presence of calf thymus DNA and 0.5M NaCl/0.008M Tris at a DNA:drug ratio of 10:1 were 1.4nm, 3.5nm and 3.6nm respectively for YCG7, YCG8 and YCG9 (ametantrone=12.2nm). Oxidative metabolism by NADPH-fortified mouse liver micro somes provided an effective route for the oxidation of acetalanthraquinones, producing two polar metabolites for YCG7 and three polar metabolites for both YCG8 and YCG9. The metabolites ofYCG7 and YCG8 were considerably more cytotoxic against the V79 Chinese hamster lung cell line than their respective acetalanthraquinones (4.4-fold for both compounds).YCG9 was found to be relatively cytotoxic per se, possibly due to its different mode of DNA binding, involving 'straddling' of the DNA helix. The increase in cytotoxicity of the metabolized products compared with the acetalanthraquinones supports the concept t?at they were conv.e~ed to their corresponding aldehydes. This leads the way to the deSIgn of acetal-contammg cytotoxic agents which can be selectively activated in tumours.

The three most likely intermediates in the mechanism of action of 5-nitroimidazole antibiotics (the radical anions of nitroimidazoles, imidazol-5-yl radicals and 5-nitroso imidazoles) have been studied. A range of 5-nitroimidazoles have been synthesised and analysed by electron spin resonance spectroscopy, (e.s.r.) and the electron spin density of their radical anions has been determined. Under reducing conditions known to proceed by a radical mechanism, a number of 5-nitroimidazoles did not undergo intramolecular cyclisation, e.g. 1-(but-3-en-1- yl)-2-methyl-5-nitroimidazolel, form stable covalent adducts with nucleotide bases, or dissociate to nitrite anions and the corresponding imidazol-5-yl radicals. This disproves the putative explanation for the generation of nitrite anions in the antimicrobial mode of action of 5- ni troimidazoles. A mechanism has been proposed to explain the release of nitrite in the mechanism of action of 5-nitroimidazoles.

It has recently been discovered that 1, 25-dihydroxyvitamin D3, the active form of vitamin D, in addition to its important role in calcium transport activity in bone, intestine and kidney, it is also found to be capable of suppressing cell proliferation and inducing cell differentiation of certain tumour cells such as malignant melanoma, breast cancer, and myeloid leukemia. The utility of 1,25- dihydroxyvitainin D3 and other vitamin D analogues as drugs in the treatment of these cancers has been restricted in part due to their potent calcemic effects. As a consequence, there has been enhanced interest in the development of structurally modified analogues of vitamin D with high cell differentiating ability and low calcemic effects. Selected hydroxylated anthrasteroids were designed to bind to the vitamin D receptors present in certain cancers, thereby inducing cell differentiation and inhibiting cell proliferation with reduction and/ or even elimination of the potent calcemic effects of vitamin D. Following a proposed scheme, the 1 (10-6) abeo-ergosta-5, 7, 9, 22- tetraen-3-one (6), a key intermediate in the synthesis of the target molecule, was synthesized starting with ergosterol. Acetylation of ergosterol gave the acetate, which was protected with 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD). Rearrangement of the adduct with BF3-ether gave the anthrasteroid-3-acetate. Hydrolysis of the acetate gave the alcohol. Oxidation of the alcohol gave 1 (10-6) abeo-ergosta-5,7,9,22-tetraen-3-one (6). The overall yield obtained over the 5-steps was 50%. After a number of unsuccessful attempts to functionalise at C-2, we were able to prepare 1(10-6) abeo-2-carbomethoxyergosta-5,7,9,22-tetraen-3-one (39), that had allowed access to our target molecules (59). Aromatisation of (39) with pyrrolidone hydrotribromide (PHT) gave the phenolic ester, followed by methylation with methyl iodide gave the methoxy ester in good yield. Reduction with lithium aluminium hydride gave the methoxy alcohol. Oxidation of the alcohol with tetra-n-propylammonium perruthenate (TPAP) gave the aldehyde in good yield. A 2-C extention via a Homer-Wadsworth- S Emmons reaction with triethylphosphonoicetate gave the side chain unsaturated ethoxy ester in moderate yield. Catalytic hydrogenation of the ester with Pd/ C gave the saturated ester in quantitative yield. Lastly, reduction of the saturated ester with lithium aluminium hydride gave 3-[1 (10-6) abeo-3- methoxyergosta-l,3,5,7,9-pentaen-2-yIJ-l-propanol (59), our target molecule in good yield. The target molecule (59) was synthesized over 14- steps with an overall yield of 10%.

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CD59 is a GPI-anchored protein found in membrane microdomains known as lipid rafts. It is a complement regulator protein, which blocks the formation of the membrane attack complex by inhibiting binding of C9 to the C5-8 complex. Human CD59 has also been described as a co-stimulator of T cell activation. The aim of this project was to analyze the role of CD59 on T cell activation in vivo. For this purpose, anti-viral CD4+ T cell responses were analyzed in mice deficient in the mouse analogue of CD59 CD59a. Infection with recombinant vaccinia virus (rVV) and influenza virus, resulted in stronger virus-specific CD4+ T cell responses in Cd59a -/- mice compared to WT mice. This effect, which indicates that CD59a downmodulates antigen-specific T cell activity, was found to be complement independent. Experiments were performed to investigate the effect of CD59 expression on human CD4+ T cells. Blocking CD59 increased proliferation of the cells in vitro indicating that CD59 might similarly downmodulate human CD4+ T cell activity. Using mouse T cells, mechanisms underlying the effect of CD59a on CD4+ T cell activity were investigated. Results of these studies indicated that downmodulation of T cell activity through CD59a requires engagement of CD59a with a ligand expressed on APCs. To assess the biological consequences of CD59a deficiency, the extent of immunopathology induced following infection with influenza virus was compared in WT and Cd59a-/- mice. Immunopathology was exacerbated in Cd59a-/- mice, correlating with increased numbers of neutrophils and CD4+ T cells in infected lungs. When complement was inhibited, lung-infiltrating neutrophils in Cd59a-/- mice were much reduced while numbers of infiltrating-CD4+ T cell remained unchanged. These results demonstrate that CD59a is more than a regulator of complement but can in fact, alter both the innate and adaptive immune responses using both complement dependent and independent mechanisms.

Cancer is a leading cause of death all over the world. Retinoic acid and vitamin D3 play an important role in cellular proliferation and differentiation and as such have potential therapeutic value as differentiating agents in the treatment of cancer and hyperkeratinising diseases. The use of differentiating agents to suppress prostate and breast cancer proliferation is now one of the new therapeutic strategies. However, the use of all trans retinoic acid (ATRA) and vitamin D3 as differentiating agents is limited by their rapid metabolism through the self induction of the cytochrome P450 enzymes that are involved in their catabolism. The P450 enzymes responsible for the metabolism of ATRA and la,25-(OH)2-D3 (calcitriol) are cytochrome P450 26 (CYP26A1) and cytochrome P450 24 (CYP24A1) respectively. Therefore the use of potent and selective inhibitors of CYP26A1 and CYP24A1 with ATRA and la,25-(OH)2-D3 respectively may represent a new strategy for the treatment of cancer. The pharmacophore model of CYP26A1 inhibitors was constructed using MOE software. A database of 71 inhibitors with different conformations have been built and arranged according to activity. The resulting pharmacophore model has 8 features of which 5 features are essential to get the CYP26A1 inhibitory activity. The designed model was used to build new inhibitors, which have reasonable activity. Two series were synthesised for CYP26A1 inhibition. The first series was designed to investigate the effect of changing the structure of the lead compound on the inhibitory activity against CYP26A1 and was depending mainly on presence of imidazole moiety, and the second series was a result of the pharmacophore search and was mainly oxadiazole derivatives. These two series were biologically evaluated using a MCF-7 breast cancer cell assay previously described by our group and also some of the compounds were tested in the biochemical assay. Changing of the structure could be tolerated to a certain limit as long as the imidazole ring is included which is the main part responsible for the binding with the haem in CYP26A1 enzyme. Some of the resulting compounds has good activity, specially methyl w-3-(1-1-imidazolyl)-3-4-(phenylamino)phenyl-2-methylpropanoate which should IC50 of 26 nM in the biochemical CYP26A1 assay. The oxadiazole derivatives did not show very good activity against CYP26A1 cell based assay, may be owing to the decrease in the.

An introduction to this work presented in this thesis involves an overview of nucleic acids and the use of nucleoside analogues in antiviral therapy. Bicyclic furano pyrimidine nucleosides (BCNA)s were discovered by the McGuigan group as potent and selective inhibitors of Varicella-Zoster Virus. A brief description of Structure Activity Relationships of this class of compounds is presented, identifying a long lipophilic chain as a specific requirements for antiviral activity. We herein report the synthesis and biological evaluation of a new series aimed to further investigate the specific requirement for biological acitivity. Two sites of BCNAs were modified on the lead compound, the side chain and the sugar moiety. A series bearing electron-donating and electron-withdrawing aryl groups was synthesised in order to investigate the role of the position of the substitutent. Then, the phosphoramidate approach was applied to the lead compound in order to broaden the spectrum of activity, which was limited only to VZV. Modifications of the sugar moiety include the inversion of all the stereo-centres of the lead compound, obtaining the L-enantiomer, and the inversion of the stereochemistry at the C-1' obtaining thus the a-derivative. Furthermore the replacement of the furano ring of the sugar with a cyclopentane was thought as a good strategy in order to increase the resistance to enzymatic cleavage. Given the poor bioavailability of the lead compound, the valyl ester was synthesised making also the hydrochloric and succinate salts to increase the chemical stability and water solubility Finally, using the intrinsic fluorescence of these derivatives, a cell study was carried out in order to investigate the distribution of the compound inside the cell.