Many important natural products contain the furan-2(5H)-one structure. The structure of this molecule lends itself to manipulation using combinatorial techniques due to the presence of more than one site for the attachment of different suhstituents. By developing different reaction schemes at the three sites available for attachment on the furan-2(5H)-one scaffold, combinatorial chemistry techniques can be employed to assemble libraries of novel furan 2(5H)-ones. These libraries can then be entered into various biological screening programmes. This approach will enable a vast diversity or compounds to be examined, in the hope or finding new biologically active Iead structures. The work in this thesis has investigated the potential that combinatorial chemistry has in the quest for new biologically active lead structures based on the furan-2(5H)-one structure. Different reactions were investigated with respect to their suitability for inclusion in a library. Once sets of reactions at the various sites had been established, the viability of these reactions in the assembly of combinatorial libraries was investigated. Purification methods were developed, and the purified products entered into suitable biological screening tests. Results from some of these tests were optimised using structure activity relationships, and the resulting products re-screened. The screening tests performed were for anticancer and antimicrobial activity, cholecystokinin (CCK-B) antagonism and anti-inflammatory activity (in the quest for novel cyclo-oxygenase (COX-2) selective non-steroidal anti-inflammatory drugs). It has been shown that many reactions undergone by the furan-2(5H)-one structure are suitable for the assembly of a combinatorial library. Investigation into the assembly of different libraries has been carried out with initial screening results included. From this work, further investigation into combinatorial library assembly and structure activity relationships of screened reaction products can be undertaken.

The metabolism of aspirin has been investigated in man following a therapeutic (600 mg) dose and after overdose. The major urinary metabolite in volunteers after the therapeutic dose was salicyluric acid and a sex-related difference in its excretion and that of salicylic acid was observed. Capacity limited formation of salicyluric acid has been demonstrated for the first time in a substantial number of paients with aspirin overdose. Urinary excretion of gentisic acid, salicylic acid and salicyl phenolic glucuronide was higher in overdose patients compared with the volunteers. In addition, the urinary excretion rates of aspirin metabolites and in particular salicyluric acid and salicyl phenolic glucuronide in individual patients varied with time after admission and the maximum observed rates were often greatly in excess of those previously reported in the literature. Depletion of plasma glycine also occurred after aspirin overdose. The in vivo plasma glycine pool was enriched by oral administration of glycine or N-glycylglycine. Glycine and N-glycylglycine treatments were as effective as the standard urine alkalinisation regimen in enhancing renal excretion of total salicylate after overdose but the mechanism of this action is unclear. Thus exogenous glycine may be of therapeutic value in salicylate overdose. The metabolism of 14c-aspirin was also studied in rats over a ten fold dose range and was found to be dose dependent. The principal urinary metabolite after each dose was salicylic acid and salicyluric acid formation was capacity limited. Oral administration of glycine concurrently with aspirin increases the urinary excretion of salicyluric acid. Gentisuric acid and salicyluric phenolic glucuronide have also been quantified in man and rat urine. Metabolism of aspirin was found to be similar in man and rat, although there were quantitative differences; thus rat may serve as a useful experimental model to study man.

The research work undertaken focused on the preparation and characterization of novel low molecular weight (LMW) hydrogels as functional biomaterials for tissue culture applications. To achieve this objective, new LMW compounds (as potential hydrogelators) were synthesized bearing a galactosamine or glucosamine moiety. The incorporation of carbohydrates was anticipated to confer molecular recognition of certain biomolecules upon the formed supramolecular gels and therefore act as potential anchor sites for cell-binding. The synthesis was based on short synthetic routes and low-cost starting materials were used as supplied. The target compounds were not confined only to those containing carbohydrates. A cinnamoyl-protected diphenylalanine hydrogelator was prepared and the properties of its corresponding hydrogel were investigated. Understanding the self-assembly mechanisms of supramolecular hydrogels is fundamental for the preparation and application of these novel materials. Therefore, a variety of techniques were employed for assessing and characterisation of gelation and to determine the configurational alignment of the formed fibres within the three-dimensional network of the gels. Specifically, the preparation and handling of hydrogels were optimized leading to robust gelation protocols. TEM and SEM microscopy revealed the size, shape and perplexing patterns of the fibres. XRD measurements verified polymorphism whereas rheology studies confirmed the viscoelastic properties of the gels. Non-covalent intermolecular interactions are the driving forces of the molecular packing, leading to higher order architectures. The combined spectroscopic analysis of the prepared hydrogels (by NMR, IR, UV-vis, CD) was advantageous to explore the nature of such interactions and allow the identification of key functional groups which actively participated in the self-assembly process. As a result of the CD work undertaken, utilisation of a synchrotron facility led to the establishment of a protocol for the evaluation of LMW hydrogels by SRCD spectroscopy, which was recently published. Finally, a preliminary biocompatibility study was undertaken to assess the toxicity of the hydrogels upon brain cancer cells. This project therefore required an interdisciplinary approach which involved the synthesis of a number of LMW compounds where some were found to be hydrogelators. This led to the preparation of their corresponding hydrogels and the study of their microscopic/macroscopic properties for the development of novel biocompatible materials suitable for tissue culture applications.

This thesis was motivated by the collaborative research undertaken by QMUL and Pfizer UK into improving experiments at pre-clinical drug development. In theory, the most efficient designs for these particular experiments, as well as for many other studies, are optimal designs. Since, however, their implementation poses challenges — and several emerged during the project — optimal designs are uncommon in practice. To address these challenges the thesis introduces a comprehensive design framework, which both generalizes and simplifies optimal design. At the core of this framework are optimal relaxed designs, seldom considered before. Like a standard design measure a relaxed design has non-integer replications and is mathematically tractable; unlike the former, whose replications must sum to one, it allows the replications total to be unconstrained. The methodology discussed in this thesis assumes design of experiments for parameter estimation, given a response model, but also applies to broader problems. Although the motivation and applications come from the pharmaceutical project, the ultimate goal is to develop an intuitive and versatile design toolkit for experimenters in various practical fields.

The aims of this project were to investigate the binding characteristics of the cytosolic oestrogen receptor in the uterus, brain and thymus of immature Wistar rats. The specificities of the receptor in the uterus are well established. The specificities of the cytosolic receptor in the uterus and thymus of immature female Wistar rats were tested against a range of steroids and the values found for the thymus compared with those for the uterus. The concentrations and dissociation constant (Kd) of the cytosolic oestrogen receptor were determined in uterus, brain and thymus of male and female rats at 5,18 and 30 days of age. Clomiphene citrate (CC), an oestrogen antagonist/partial agonist, oestradiol (E2), CC+E2 or 4-hydroxyandrostenedione (4-OHA), an aromatase inhibitor, were administered to animals at 15 days in order to study the effects of these compounds on receptor binding characteristics at 30 days. Significant differences in specificity were found between the thymus and uterus, the cytosolic oestrogen receptor in the thymus exhibited significantly higher affinity for corticosterone than it did in the uterus. Male animals were heavier than females at 30 days. Uterus and thymus weights increased exponentially between 5 and 30 days. The tissue-to-body weight ratio increased in uterus between 18 and 30 days and increased in the thymus in both sexes between 5 and 18 days. In males at 30 days, the tissue-to-body weight ratio of the thymus was significantly lower than in females of the same age. Cytosolic oestrogen receptor concentrations in the uterus, brain and thymus differed between some age and/or sex groups. Cytosolic oestrogen receptor concentrations increased exponentially in the uterus between the different age groups. Cytosolic oestrogen receptor concentrations in both thymus and hypothalamus at 5 days were significantly higher in females than in males of the same age group. No differences in cytosolic oestrogen receptor concentrations were found between the sexes in the cortex at 5 and 18 days but at 30 days, receptors were not detectable in this brain area. The Kd for moxestrol, a synthetic oestrogen agonist that is not bound by alphafetoprotein present in the blood of immature rats, was similar in all tissues. E2 and CC+E'-' treatmentsr esulted in decreasedb ody and thymus weight in both sexes,i ncreased uterus weight and decreased thymus weight in both sexes but led to increased uterus weight. CC treatment decreased the concentration of the receptors in the female thymus only-, E2 and CC+E2 treatments decreased the concentratIon of the receptor to levels that were undetectable in hypothalamus and thymus in both sexes, 4-OHA treatment increased thymus weight and cytosolic receptor concentrations in the hypothalamus and thymus of males only. These results suggest that cytosolic oestrogen receptors in uterus, brain and thymus are similar and that sex differences in these tissues are mediated by differential exposure to oestradiol during the early postnatal period. The thymus is crucial to the development of the immune response. The finding that the cytosolic oestrogen receptor differed from the uterus receptor in its affinity for corticosterone and that sex differences in cytosolic oestrogen receptor concentrations were present in the thymus at 5 days could be relevant to the sex dimorphisms that exist in autoirnmune disease manifestation.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease in the growing population of elderly people. Although the etiology of the disease is yet to be fully elucidated, pathological hallmarks have been consistently described, including the accumulation of amyloid plaques, dysfunctional ionic homeostasis, synaptic disruption and neurodegeneration. The amyloid hypothesis postulates that aberrant production of amyloid-β (Aβ) proteins, which have a high propensity to aggregate, lies at the center of the pathological mechanism of AD. In particular, soluble oligomeric Aβ structures have been identified as primary toxic species. The interaction of these structures with several cellular targets, including ion channels such as nicotinic acetylcholine receptors (nAChR) and voltage operated Ca²⁺ channels (VOCC), has also been implicated in Aβ toxicity and AD. The aim of this thesis is to investigate how the acute and chronic actions of Aβ in vitro are affected by nicotinic ligands. Acute application of Aβ₁₋₄₂ to fluo-3-loaded PC12 cells potentiated Ca²₊ increases evoked by stimulation of nAChR and VOCC, while chronic application reduced redox potential, disrupted membrane integrity and initiated apoptosis in PC12 cells. In addition to mimicking the toxic responses of PC12 cells, Aβ₁₋₄₂ also reduced neurite outgrowth and synaptogenesis in rat primary cortical neurons. All actions of Aβ were prevented by inhibitors of Aβ₁₋₄₂ oligomerisation, including the hexapeptide KLVFFA. Neuroprotection afforded by (+)-nicotine also occurred via inhibition of Aβ₁₋₄₂ oligomerisation, rather than by a receptor-mediated mechanism. No other pharmacological approaches, including application of two novel ligands selective for α7 nAChR: the partial agonist SSR180711 and antagonist α-conotoxinArIB[V11L,V16D], characterized herein, protected against Aβ₁₋₄₂ toxicity. While inhibiting oligomerisation prevented the actions of Aβ₁₋₄₂, enhanced oligomerisation evoked amplified toxic responses. However, the potentiation of Ca²⁺ signalling diminished following enhanced oligomerisation. This, coupled with a lack of VOCC-involvement in Aβ toxicity and the differential actions of truncated Aβ peptides on toxicity and Ca²⁺ signaling, indicates that the acute disruption of Ca²⁺ signaling by Aβ does not underpin the chronic toxic effects of Aβ.

Chitin, a linear polymer of N-acetylglucosamine, is an essential structural component of fungal, nematode and insect pathogens but is not found in human physiology. Chitinases, which hydrolyze this polymer, play a key role in life cycle of these pathogens and associated pathogenesis. Consequently chitinase inhibitors have generated a lot of interest given their potential as insectides, fungicides and antimalarials. Herein, approaches are reported to the synthesis of some non-sugar based chitinase inhibitors: the cyclic pentapeptide natural products argifin, banyasin A and diketopiperazines related to the natural product CI-4. In order to improve the efficiency of production of argifin and facilitate SAR on analogues of the natural product, a revised synthesis of argifin has been developed. The synthesis of argifin was carried out by a combination of solid-phase and solution chemistry. The assembly of the linear peptide was carried out by SPPS and the cyclisation was performed in solution. The protecting groups chosen for the Asp and Arg residues were removable by hydrogenolysis, as this allowed aspartimide formation under acidic conditions to be avoided. Only one HPLC purification was required at the final step; argifin was isolated in 19% yield, compared to 18% yield for the first synthesis by Dixon et al. Banyasin A contains the same essential Arg(MC)-MePhe dipeptide motif as argifin and so it is of considerable interest as a potential Family 18 chitinase inhibitor. The synthesis of Amoa (3-amino-2-methyl-5E-octenoic acid) a rare amino-acid present in banyasin A was investigated. An advanced intermediate for the synthesis of Amoa was successfully obtained via chiral pool chemistry in an 8 step sequence from L-Asp. This involved preparation of a selectively protected β-methyl-substituted Asp derivative, which was then homologated to the β-amino-acid via Arndt-Eistert chemistry to give (3R,4R)-3-(((benzyloxy)carbonyl)amino)-4-methyl-5-oxo-5-allyloxypentanoic acid in 27% yield for the final step. The cyclic dipeptide CI-4, cyclo (L-Arg-D-Pro) is a weak inhibitor (IC50 = 1.2 mM) of Family 18 chitinases, however its binding efficiency index (BEI) is comparable to more potent inhibitors such as argifin. Some analogues of CI-4 show promising activity against a typical bacterial type Family 18 chitinase, SmChiB1 from Serratia marcescens. The cyclic dipeptide should therefore be a useful starting point for the development of more effective and selective inhibitors of this enzyme class. A series of cyclo (Xaa-Pro)-based dipeptides were synthesized, with different Xaa such as L/D-Pro, Gly, L-Ser, L/D-Arg, D-His, D-Phe, with yields ranging from 12 to 84%. Preliminary biological data confirm that cyclo(D-Xaa-D-Pro) may be a novel template for the development of new drug-like inhibitors of Family 18 chitinases.

The aim of this research was to develop a new work model for hospital pharmacists based on risk assessment of patients using an electronic prescribing and administration system (EPMA). Systematic review was performed to identify risk factors associated with clinical pharmacy intervention. Those factors which can be measured by the EPMA in a UK teaching hospital were subsequently identified. Data was extracted from the EPMA relating to risks in intervention recipients on medical and surgical wards and those patients present concurrently. Univariable and multivariable analysis was performed and a risk score calculated. Receiver operating curves (ROCs) determined predictability of the score. Risk factors for pharmacist intervention were: age, female gender, patient compliance, unavailable stock, prescription of warfarin, number of allergies, comorbidities, regular prescriptions, anti-epileptics, thrombolytics/anticoagulants, central nervous system agents, and chemotherapy / immunosuppressants. The area under the ROC for the risk score was 0.61. Multiple factors were significantly and independently associated, with an increased intervention rate. However, it was not possible to generate a useful model for directing clinical pharmacy services. Inverse relationships were demonstrated between some risk factors usually associated with problems with medicines use.

The demand for novel antibiotics in the treatment of infections by \(Mycobacterium\) \(tuberculosis\) (\(M.\) \(tb\)), the causative agent of tuberculosis (TB) has reached new highs with the recent emergence of totally-drug resistant TB (TDR-TB). Efforts to develop antibiotics with revolutionary mechanisms of action, low minimal inhibitory concentrations (MICs) that will decrease the current drug burden and clear infection without compromising side effects has led to several collaborative efforts between pharmaceutical companies and academic institutions throughout the world. This interdisciplinary collaborative effort and advances in drug development technologies, such as high throughput phenotypic screening of compound libraries and whole genome sequencing (WGS) has accelerated the identification of new compounds (or hits) with potent anti-tubercular activity and delivered a variety of unique drug targets. These hits are also potent against drug-resistant strains of \(M.\) \(tb\), and have significant efficacy in vivo models of TB infection. Bedaquiline (Sirturo™) is the first new specifically designed anti-TB drug to be approved by the Food and Drug Administration (FDA) for use against multi-drug resistant (MDR-TB) in 40 years. This considerable step forward marks the beginning of a paradigm shift in antibiotic development, signaling a new age of drug discovery. In this thesis, the modern age of TB drug discovery will be examined as well as the efforts made to further this field through the identification of novel hits with activity against \(M.\) \(tb\) and identifying their respective inhibitory mechanisms.

Nucleoside analogues are an important class of antimetabolites, used both as anticancer and antiviral agents for their resemblance to endogenous nucleosides, and their capacity to inhibit metabolic pathways in which these substrates are involved, leading to therapeutic potential. The need for both new anticancer and antiviral cures is significant, and often caused by the emergence of resistance to the available therapies. In the case of therapies with nucleoside analogues, the delivery of a nucleoside monophosphate prodrug inside the cell has potential to overcome resistance to treatment, and proved successful especially with the application of the ProTide approach. This strategy led to the progression into clinical trials of numerous antiviral and anticancer agents. This work focused on the synthesis of novel ProTide prodrugs to different nucleoside analogues that are involved in clinical trials, such as purine and pyrimidine nucleosides with modifications in the base and sugar regions. This strategy was also applied to nucleoside analogues where in vitro evaluation has never been reported. The synthetic strategies to prepare each nucleoside analogue are also reported. The in vitro evaluation of novel nucleotide prodrugs as anticancer and antiviral agents is described and discussed. Moreover the mechanism of activation of the ProTides is supported by studying their bioactivation to the corresponding monophosphate forms, through enzymatic NMR studies and molecular modeling simulations. Modifications on the scaffold of the two most promising families of ProTides were also performed, leading to the introduction of groups on the nucleobase or alteration of the sugar moiety. Moreover, the phosphosphate group was also modified, with the application of alternative phosphorodiamidate and phosphonoamidate prodrug approaches, with the aim of improving the biological profile. Selected analogues from two families were submitted to numerous preclinical assessments, retaining better potency compared to the parent nucleosides. Moreover these analogues were stable in human plasma, serum and liver microsomes. Further investigations on these potential new drugs are currently ongoing.