There are many strategies used in high-throughput-synthesis and one technique, based on the use of polymer-supported reagents in solution-phase, has evolved considerably over the past decade. However, only a limited number of efficient polymer-supported coupling reagents have been reported and many of them offer serious drawbacks, unlike their solution-phase equivalents. A comparison between common coupling reagents (IIDQ, HATU, PyAOP and BOP-CI) was therefore carried out and revealed that IIDGQ was the reagent of choice in the absence of the activation step. Polymer-supported IIDQ was therefore targeted.  This reagent performed better than commercially available HATU and all supported carbodiimides. Scope and limitation studies proved that the reagent was a versatile tool for the synthesis of amides from carboxylic acids and amines, including hindered substrates, secondary amines and anilines. A new strategy for the high-throughput-screening of small-molecules was developed. Microarrays are a powerful method to test quickly a wide range of small molecules against biological targets. In parallel, fragment-based drug discovery has boomed in the past decade. The combination of the two principles gave birth to the new concept of dual fragment microarrays. The technology was developed and evaluated using fragments known to bind to the human FK506-binding protein. These fragments were synthesised in solution-phase, then linked to a PNA tag synthesised on solid-phase, followed by release into stock solution and hybridisation onto DNA microarrays. Screening the arrays of mixtures of two fragments revealed that the expected strongest “dual” interaction was detected as the most intense spot, and thus validated the concept of dual fragment microarrays.

This thesis is concerned with the design of ruthenium(II) arene anticancer complexes where the chelating ligand contains an azo-imine group (­N=N-C=N) which binds to the ruthenium to form a five-membered chelate ring. Several mononuclear complexes containing the chelating 2-phenylazopyridine ligand or derivatives thereof were found to be moderately cytotoxic towards A2780 human ovarian and A549 human lung cancer cells (IC₅₀ 18-88 μM). These complexes were found to hydrolase (Ru-C1 → Ru-OH₂) slowly in aqueous solution and arene loss was a competing reaction. X-ray crystal structures revealed that the arene ligand is not as tightly bound to the ruthenium as in the case when the chelating ligand is en. Synthesis of the corresponding dinuclear analogues, where two 2-phenylazopyridine ligands were joined together via a linker, did not improve the cytotoxicity. Unlike their mononuclear analogues, the complexes appeared to undergo electrochemical reduction with radical formation in aqueous solution. An EPR spectrum of the mono-reduced anion revealed an entirely ligand based radical. Surprisingly, replacement of Cl^- by I^- in the mononuclear complexes led to compounds that were highly cytotoxic towards both A2780 and A549 cancer cells (IC₅₀ 1-5 μM). Interestingly these complexes were resistant to hydrolysis in aqueous solution suggesting that this class of compounds had a novel mechanism of cytotoxic action not involving activation by hydrolysis. The redox chemistry of these complexes proved to be central to their observed cytotoxicity. The complexes were rapidly reduced by the biological reductant ascorbate, catalytically oxidised the tripeptide glutathione and generated reactive oxygen species (radicals) inside A549 cancer cells. These reactive oxygen species were shown to be involved in cell death. These iodide complexes were also found to be cytotoxic towards non-cancerous WI38 human lung cells (IC₅₀ 1-6 μM) and attempts were made to target one complex specifically to cancer cells, through conjugation to transferrin.

A solid-supported phosphorylation reagent was developed, based on Ludwig-Eckstein chemistry. Different resin supports were evaluated and microporous polystyrene with 1% crosslinking was found to give the best results. The supported reagent was characterised by ³¹P Gel-phase NMR and phosphorous content and applied to the synthesis of DNA triphosphates. The triphosphates were isolated in 28-50% yields, often without the need for ion exchange chromatography, using preparative HPLC for purification. Propargylamino linked triphosphates were also prepared using this approach. The reagent was successfully applied to the synthesis of a fluorescein labelled Thymidine triphosphate. Using the supported reagent, complete phosphitylation of the alcohol could be demonstrated. The product was isolated by preparative HPLC and its identity proven by ³¹P NMR and mass spectrometry. Additionally, the reagent’s potential as a handle to access different phosphorylated compounds, such as mono-<i>H</i>-phosphonate esters, phosphate diesters, phosphate trimesters and diphosphoramidates was explored and its application to the synthesis of nucleoside prodrugs is discussed.

Photoactive platinum compounds have the potential to reduce some of the debilitating side-effects associated with conventional chemotherapeutics, such as cisplatin. Stable, inert platinum(IV) compounds which are reduced to active platinum(II) species only upon irradiation, could provide a site-specific treatment. The Pt^IV azide complexes, <i>cis, trans, cis-</i>[Pt(N₃)₂(OH)₂(NH₃)₂] and <i>cis, trans-</i>[Pt(en)(N₃)₂, have previously been shown to be stable in the dark but reduced to Pt^II upon irradiation. The synthesis and characterisation of new platinum azide compounds, designed to improve important properties such as solubility and wavelength of absorbance are described here. Complexes which have azide ligands in a <i>trans </i>position were synthesised, the general formula is <i>trans, trans, trans</i>-[Pt(N₃)₂(OH)₂(NH₃)R] where R is NH₃, pyridine, methylamine, ethylamine, thiazole, 2-picoline, 3-picoline, 4-picoline or cyclohexylamine. Several Pt^IVdiazido compounds containing chelating aromatic ligands, such as 2,2’-bipyridine and 1,10-phenanthroline were also prepared. Many of the novel compounds synthesised were characterised by X-ray structure determination. The complexes with <i>trans</i> azides generally showed improved water solubility as well as a shift of the main absorbance band towards the visible region, compared to their <i>cis </i>analogues. A transcription mapping study of a fragment of pSP73KB plasmid DNA treated with <i>cis, trans</i>-[Pt(en)(N₃)₂(OH)₂] and visible light, has shown that platination mainly occurs at consecutive guanine bases. The major binding sites were similar to those of cisplatin. No platination was seen in an identical sample which was not irradiated.

The disruption of the p53-Mdm2 interaction is an attractive therapeutic strategy for activating p53 tumour suppressor activity in tumours expressing wild type p53. One such strategy for the disruption of the P53-Mdm2 interaction is the design of low molecular weight ligands that bind at the p53-Mdm2 interface. The molecular docking programme LIDEAUS has identified a sulphonamide scaffold that has the potential to bind at the p53-Mdm2 interface. Adopting a high-throughput, combinatorial chemistry approach, a number of sulfonamides and amide have been synthesized and screened <i>in vitro</i> for their ability to disrupt the p53-Mdm2 interaction. Cyclin dependent kinases (CDKs) are important in cell cycle regulation as they control the transition between the four primary phases. The association of a cyclin dependent kinase with a cyclin subunit, and subsequent phosphorylation of the complex formed, is essential for CDK activity. Therefore, the design of ATP antagonists that inhibit the activity of CDKs is an attractive strategy for the treatment of proliferative disorders. Using microwave assisted organic synthesis, a high-throughput synthetic sequence has been developed to afford a series of 6-anilino-4-aryl-pyrimidines (I) that have been subsequently screened <i>in vitro</i> against a panel of kinases. Several of these compounds have been shown to be low micromolar inhibitors of the CDK2/cyclin E complex.

There have been many approaches taken in drug delivery aimed at improving the efficacy of active agents. The way in which drugs are administered could be a key factor for an optimum treatment and hence the delivery system used may be as important as the active drug. The aim of this thesis was to investigate the use of embolic micro spheres as an effective drug delivery system, for treatment of malignant tumours including primary and secondary liver cancer, and benign tumours such as uterine fibroids. Embolisation is a treatment for destroying tumours and other growths found in the body by blocking their blood supply. Chemoembolisation uses a combination of delivered drug and embolisation to provide specific targeting of the drug at the site of the tumour and has been used successfully to treat hypervascular tumours such as hepatocellular carcinoma One of the most challenging aspects of the embolisation procedure is effective post-procedural pain management. Biocompatibles UK Ltd has developed two poly(vinyl alcohol) based nondegradable embolic micro spheres, GelSpheres™ and Bead Block™, This study has demonstrated that GelSpheres have the ability to actively load and release cationic drugs such as doxorubicin and irinotecan in a controlled fashion via an ion-exchange mechanism. The loading and release of both drugs from the GelSphere showed no detrimental effect on the micro spheres in terms of morphology, deliverability and handling or on the stability of the drugs. The loading and release kinetics of both drugs are described together with in vitro-in vivo correlations for these products. In addition, a method was developed to load Bead Block with a non steroidal anti-inflammatory drug (NSAID), ibuprofen. This system demonstrated sustained release of the ibuprofen and the potential to be used as an embolic agent to control inflammation and pain. These drug delivery systems could provide an alternative to conventional embolisation and chemoembolisation procedures, providing ideal formulations for local and sustained delivery of drugs following embolisation.

Background and aims Intrathecal drug delivery (IDD) is a last resort treatment for the management of severe chronic pain due to its invasive nature, high initial cost, concerns about long-term opioid use and possible complications related to the procedure. Although it has been available since 1981 some of the possible complications of this treatment have only recently been observed and are not fully understood. Additionally the current available literature exploring the longterm effectiveness and cost effectiveness of IDD is limited. The aims of this study were to explore some of the least investigated complications, long-term effectiveness and cost effectiveness of IDD. Methods The methodology used included both retrospective and prospective data collection to investigate intrathecal morphine dose escalation, granuloma formation, hormonal effects, bone mineral density (BMD), long-term effectiveness and cost effectiveness of IDD. Results A model was developed that allows the prediction of the intrathecal opioid dose at year six of therapy based on year two dose and the duration of pain prior to initiation of intrathecal therapy. An association between opioid concentration and formation of intrathecal granulomas was confirmed for the first time in humans and a tool to assist recognition of asymptomatic granulomas was identified. It was observed that hypogonadism is prevalent in this population and free testosterone is a more reliable method to diagnose this condition. An association between low BMD and hypogonadism as a result of IDD was reported for the first time. Effectiveness of IDD was verified following a mean of 13.5 years of therapy. A new concept with implications for cost analyses was identified. Conclusion This study presents a thorough analysis of IDD therapy. Despite potential side effects, this therapy can be effective over periods longer than 10 years in appropriately selected patients. The cost effectiveness of IDD systems was confirmed based on real patients’ data. Most side effects can be prevented with attentive follow-ups.

Since ancient times, substances derived from amphibian skins have been recognised to possess various medicinal properties, In recent years, the peptides from amphibian skin have attracted extensive attention for their profound significance in providing clues directed toward novel drug development, for better understanding of miscellaneous physiological and pathological processes, for elucidation of phylogenetic relationships and for improving taxonomy. Previously, the chemical complexity of the defensive skin secretion of the broad-folded frog, Hylarana latouchii, has not been studied in detail. In this thesis, parallel transcriptomic and peptidomic analyses of the electrically-stimulated skin secretion have enabled the identification and characterisation of seven, biologically-active peptides. Five of these peptides are antimicrobial peptides displaying differential growth-inhibitory activity toward test microorganisms and human cancer cell lines; two of which belong to the previously-identified brevinin-l and temp orin families, while the others show little structural similarity with other antimicrobial peptides and represent the prototypes of a novel peptide family - the hylaranins. Hylaranin-Ll and hylaranin-L2 (two 18-mer structurally-related but distinct peptides) are described in Chapter 3 and hylaranin-L3 (a unique l3-residue peptide) is described in Chapter 5. In Chapter 4, a novel Bowman-Birk-type trypsin inhibitory peptide, pLR-HL, belonging to the pLRlranacyclin family, was identified. By substitution of its Lys8 residue with Phe, the Phe8-pLR-HL analogue was found to be transformed into a chymotrypsin inhibitor. The II -residue canonical reactive loops within each peptide could exert corresponding protease inhibitory activity independently, In Chapter 6, a peptide structurally-related to bombesin was isolated and found to possess contractile activity on rat urinary bladder and uterus smooth muscles. These data illustrate that amphibian skin continues to provide numerous novel peptides for furthering research in the fields of pharmaceutical science, biological chemistry, medical science, and systematics. It is expected that many more novel amphibian skin peptides are awaiting discovery.

No description available.

Antibiotic resistance arising from the selective pressure generated by excessive/inappropriate antibiotic use in human and veterinary practices poses major challenges to the management of infection, particularly with the scarcity of new antibacterial drugs. For this reason, there is considerable interest in developing strategies to counteract multidrug microbial resistance either as an independent pharmaceutical entity or as an adjunct to existing treatment regimes. Cyclooygenase (COX) metabolises phospholipase A2-liberated arachidonic acid to PGH2, which serves as a substrate for down-stream synthases to generate prostaglandins and thromboxane A2. Two isoforms of COX exist with constitutively expressed COX 1 suggested to make PGs to aid physiological processes while COX 2 is inducible at sites of inflammation believed to generate pathophysiological PGs. During inflammation in response to infection, PGs of the E/D series elevate cAMP by activating EP2/EP4 or DP1 receptors, respectively. Elevating cAMP inhibits two pivotal steps in NADPH oxidase-mediated bacterial killing, namely the phosphorylation as well as the translocation of the cytosolic p47phox subunit to cell membrane. Moreover, by signaling through EP2/4 PGE2 inhibits FcyR-mediated phagocytosis. As non-steroidal anti-inflammatory drug (NSAIDs) classically inhibit PG synthesis, it is not surprising that NSAlDs are increasingly recognised to facilitate leukocyte killing of bacteria. That notwithstanding, properties of COX 1 versus COX 2 inhibitors is unknown as is their respective roles in PG synthesis, cAMP expression and therefore cytokine balance during infection in both mouse and humans. Moreover, it is not known whether NSAlDs interfere with antibiotic-mediated bacterial killing. Finally, it is unknown whether priming the innate response by PG inhibition would enhance leukocyte killing of antibiotic-resistant bacteria by overcoming strategies drug-resistant bacteria have developed to parry antibiotic efficacy. To investigate this, we carried out a series or experiments in mouse and in humans finding that COX 1 is the predominant isoform active in PG synthesis during infection and that prophylactic as well as therapeutic inhibition of both COX isoforms kills bacteria to an equivalent extent by increasing phagocytic uptake and reactive oxygen intermediate-mediated killing. Moreover, we report that inhibition of PGs synthesis and signaling enhances bacterial killing in humans; that NSAlDs do not interfere with the mode of action of antibiotics but exert an additive effect when used in combination with penicillin, for instance. Finally, we show that priming the innate immune system with NSAIDs bypasses antibiotic resistance and kills drug-resistant bacteria. These data underlie the importance of lipid mediators in host responses to infection, the potential of inhibitors of PG signaling pathways as adjunctive therapies, particularly in the context of antibiotic resistance.