Vibralactone is a potent inhibitor of pancreatic lipase and a potential candidate for a new anti-obesity therapeutic. Not only is vibralactone an interesting molecule due to its topical biological activity, but its unique fused bicyclic B-lactone structure also presents a challenging target for synthetic investigation. Herein two novel and highly diastereoselective total syntheses of (±)-vibralactone will be presented along with efforts towards an asymmetric synthesis of (-)-vibralactone. Highly functionalised intermediates have been accessed by a unique titanium-induced acetal addition to malonate precursors, followed by a highly diastereoselective allylation to set the relative stereochemistry of the adjacent chiral centres of vibralactone. Two synthetic routes to vibralactone have been completed from these novel scaffolds in 17 and 11 steps with overall yields of 4.5% and 16% respectively. Both routes exploit aldol condensations to prepare the cyclopentene ring of vibralactone, and the second-generation synthesis employs a novel and very efficient deallylation/cyclisation reaction to form the B-lactone ring. Two all-trans-retinals ([10-18-13C9] and [12,15-13C2]) containing multiple 13C labels have been synthesised to facilitate DNP enhanced solid-state MAS NMR investigations into the ring orientation and retinal interaction with transmembrane retinylidene proteins, proteorhodopsin, channelrhodopsin and KR2. Channelrhodopsin and KR2 have very important applications in optogenetics; a technique that can be used to probe how the brain functions and has the potential to relieve symptoms of neurological conditions. The labelled retinal isotopomers have been accessed from simple and readily available 13C-enriched starting materials using a synthetic route that will ultimately permit access to other labelling patterns as required.

Due to the physical limitations associated with the size of a hard gelatin capsule shell it has not been feasible in practice to present a low potency drug in the form of a sustained release multiparticulate delivery system. The aim of this work was to design a tablet which, on oral administration, rapidly releases intact polymer coated pellets in which the integrity of the cores and the release retarding membrane is preserved. A comprehensive study is made of the effect of uncoated pellet formulation and pelletization processing variables on the physical, skeletal and tensile properties of uncoated pellets. Investigation into the effect of the drying technique on the properties of uncoated pellets is also reported. This work has shown that the aqueous solubility of pellet components and the drying technique used has a marked effect on pellet diametral strength, elasticity, surface characteristics and in-vitro drug release. Pellets were coated using aqueous dispersions of polymethacrylate, ethylcellulose and silicone elastomer polymers. The effect of the film composition on tensile properties was studied by evaluating the mechanical properties of free-films using the technique of indentation hardness testing. The quality of the film coating and the effect of polymer loading was evaluated using in-vitro dissolution testing and by scanning electron microscopy. It was found that those polymeric film formulations whose tensile properties most resembled the tensile properties of the pellet cores resulted in films which were best able to withstand the applied stress associated with pellet compaction. Those films exhibiting significantly greater elasticity than the uncoated cores resulted in pellets which exhibited a tendency for instantaneous elastic recovery on removal of the applied load; tablet formation was therefore prohibited. Polymer coated pellets were successfully compacted into tablets with an inert direct compression blend comprising large particle size grades of lactose and microcrystalline cellulose. Pellet distribution within the tablet matrix (evaluated by image analysis of tablet sections, microphotography and uniformity of content data) failed to show evidence of particle segregation. Comparative in-vitro release profiles of compacted and non-compacted pellets shows that some physical damage is being caused to pellets as a result of the compaction process. Pellets are rapidly released from the matrix on disintegration of the tablet. Visual observation of released pellets indicated that they were intact, however microscopic examination revealed evidence of impaired surface quality. Pellet damage during compaction appears to be independent of the magnitude of the applied load and fracture of the polymer coating is restricted to those pellets in contact with the surfaces of the punches and die during tableting.

No description available.

A study has been conducted aimed at the generation and characterisation of mutations in the Escherichia coli gyrA gene, resistant to the quinolone group of antibacterial agents. Preliminary studies on quinolone-resistant mutants of strains that over-express the DNA gyrA gene, revealed the over-production of a 60 KDa protein which was partially purified. This 60 KDa protein was found to be similar, but not identical to the E. coli heat shock protein GroEL. The gyrA gene has been recloned in the 8.0 kb plasmid pPH3, which contains the gene under the stringent control of the hybrid tac promoter. The E. coli strain JMtacA containing pPH3 exhibits no expression of the gyrA gene in the absence of the inducer (IPTG), but over-produces the protein at greater than 20% of the total soluble cell protein after induction. The optimisation and purification of the GyrA subunit from JMtacA is also described. A fragment was subjected to site-directed mutagenesis which contained the TCG codon for serine-83, which was mutated to alanine (GCG). The mutant showed a 15x increase in MIC50 compared to wild-type. The mutated GyrA subunit was over-produced, complexed with wild-type GyrB subunit and used in various assays for reactions performed by DNA gyrase. The ID50 was determined for supercoiling, decatenation, relaxation of negatively supercoiled DNA, and cleavage of supercoiled DNA. The cleavage reaction mediated by Ca++ was also investigated. The technique of gap-misrepair mutagenesis, geared to the generation of single, random point mutations on a plasmid was also used on the plasmid pPH3, to generate a quinolone-resistant mutant of the gyrA gene. The mutant isolated (GMIOO) was also over-produced and compared to wild-type in various assays. The mutation was determined by DNA sequencing to be glutamine-106 to arginine.

Photodynamic therapy (PDT) is a novel treatment for malignant disease. The first step is intravenous injection of a light-absorbing, cytotoxic drug (the photosensitizer) that is then allowed time to accumulate in malignant tissue. The second step involves local activation of the photosensitizer with long (red) wavelength light, delivered usually from a laser. Subsequent to irradiation, highly reactive singlet molecular oxygen (Type II mechanism) is likely to be the most damaging cytotoxic species in vivo. The porphyrin molecule, Photofrin®, is the only photosensitizer currently registered for clinical use but is associated with several problems. Most disappointing is the fact that Photofrin® accumulates not only in malignant tissue but also in other organs, such as the liver, kidney and spleen. Its long persistence in the skin commonly causes severe photosensitization reactions in patients for up to three months post-treatment. Photofrin® also has poor light absorption properties within the therapeutic window (600 to 800 nm) for PDT. Furthermore, PDT with Photofrin® has proved of no use in the treatment of malignant melanoma due, possibly, to competition between the photosensitizer and melanin for light absorption. Second-generation photosensitizers have tended to be porphyrin-based molecules, many of which have reached various stages of clinical trial. Of non porphyrin-based compounds, the cationic dye, methylene blue (MB), used traditionally as a nuclear stain in histology, has proved also to be an efficient photosensitizer, with maximum light absorption properties within the therapeutic window for PDT. Its use as a selective stain for tumour tissue in the bladder led first to its investigation in humans for the PDT of bladder cancer and inoperable tumours of the oesophagus. Radiolabelled MB has also recently been used as a tracer for metastatic melanoma in humans. The disadvantages of MB are an inherent (dark) toxicity and its rapid reduction in vivo to the inactive form, leuco-methylene blue (LMIB). This study found the cytotoxicity of MB to be enhanced by illumination and that successive methylation of the molecule corresponds to both increased light and dark toxicities in the EMT-6 (murine mammary), the SK-23 (murine melanoma) and SKMEL-28 (human melanoma) cell lines. The increased toxicities may be due to increased resistance to reduction (MBcNMB.cMMB < DMMB), improved singlet oxygen quantum yield (MB.cMMBcDMMBcNMB), increased hydrophobicity (MB.cMMBCDMMBcNMB), improved cellular uptake (MBctvHv1BcDMMBNTvffi) and/or changes in intracellular targeting and localisation. MB is known to target the nucleus but it was proposed that the increased hydrophobicities could lead to the mitochondrial targeting of the derivatives. The intracellular localisation of the photosensitizers following incubation was studied using both fluorescence and confocal microscopy. Here, confocal microscopy showed that none of the four photosensitizers, including MB itself, target the nucleus prior to illumination. DMMB and NIMB in particular appear to be confined to small subcellular bodies within the cytoplasm. However, the precise locations of the photosensitizers, prior to illumination, were not established during the course of this study. Nevertheless, confocal microscopy showedthat, upon illumination, all four photosensitizers rapidly relocalise within the nucleus. Since photosensitizers that localise in mitochondria are found to be more efficient inducers of apoptosis than photosensitizers that target other subcellular sites, the ability of the photosensitizers, MB, MMB, DMMB and NMB, to induce apoptosis in EMT-6, SK-23 and SK-MEL-28 cells in culture was investigated in this study. The methods used were visual examination of cell morphology, by use of the cyanine dye, JC-1, and the use of the FluorAce® Apopain Assay Kit from Biorad, in cells that had been exposed to the photosensitizers. From these, it was concluded that an apoptotic cell killing mechanism might play an important role in the photocytotoxicity of the photosensitizers, moreso for DMMB and NMB. The overall purpose of this project was to assess the potential of the derivatives of MB to be used in the PDT of cancer, with a special emphasis on malignant melanoma, since this is a field of cancer treatment where PDT has had no success. Although methyl substitution of MB did not abolish the inherent toxicity of the molecule, it is possible to assess the potential usefulness of the photosensitizers by examination of the light: dark differential. In fact, the light:dark differential was improved for all the derivatives of MB in all three cell lines. Nevertheless, NMB consistently performed the best, achieving maximum photocytotoxicity at concentrations that caused very little toxicity in the dark. The presence of melanin made no difference to the photosensitizing capabilities of the photosensitizers in melanoma cells. In terms of a clinical application of the current work, PDT employing phenothiazinium photosensitizers is not suggested procedurally for the removal of primary melanoma, since this is routinely performed by excision. However, due to the demonstrated efficacy of MB in tracing microsatellites and its use in sentinel lymph node tracing, it may be of use in the photodynamic treatment of local metastatic lymph infiltration immediately post-surgery, as an alternative to lymphadenectomy. At present, MB is used routinely in various tracing or demarcation procedures, either visible or scintillographic, without reported toxicity. The derivatives used in the present in vitro study were all more effective in terms of the photodynamic effect and it is thus possible that future clinical developments in this direction may be feasible.

No description available.

No description available.

A high proportion of prostate cancer has been shown to be hormone-dependent, in particular, dependent on testosterone (T) and dihydrotestosterone (OHT). The biosynthesis of the androgens is catalysed by the cytochrome P-450 enzyme 17a.-hydroxylase/17,20-lyase (P45017a) which is responsible for the conversion of pregnanes (for example progesterone) to . the androgens (for example androstenedione). However, the biosynthesis of OHT, the more potent androgen is catalysed by 5a-reductase (5aR). The inhibition of these two enzymes would therefore lead to the overall reduction in the level of T and OHT. Thereby leading to a decrease in the stimulation of androgen-dependent cancer cells. Within the current study, the synthesis of a series of potential inhibitors is described. The compounds synthesised against P45017ó were based upon the ability of the compounds to donate a lone pair of electrons to the iron atom within the haem group of the active site of P45017a. As such, compounds based on the Evan's chiral auxiliary were synthesised containing a phenylamine moiety. In the synthesis of the compounds, the initial R and S forms of the chiral auxiliary were initially alkylated (using sodium hydride and alkyl bromlde), followed by the nitration (with dilute fuming nitric acid) of the phenyl ring which was subsequently reduced (using palladium on charcoal and hydrogen gas) to give the phenylamine moiety. The reactions proceeded in moderate to good yield without many problems. The reactions were repeated with an alternative chiral auxiliary, namely, 4-methyl-5-phenyl-2- oxazolidinone, however, due to lack of time, only the initial alkylation step was undertaken and was found to proceed in good yield. The biochemical evaluation of the phenylamine based compounds using a literature based assay showed these compounds to be weak inhibitors of P45017a, with two compounds (of the range evaluated) showing close to 40% inhibition at 1mM. The compounds targeted against 5aR, were based upon the ability of the _inhibitors to mimic the substrate and thereby allow themselves to be reduced by NAOPH, as such, they were based upon pyrrolidine-2,5-dione structure with a C=O moiety to mimic the 11[sup]5 functionality in T. The N-substituted pyrrolidine-2,5-dione was therefore reacted with the appropriate ester in the presence of sodium hydride. In general, the reactions proceeded well, however, the products were obtained in poor yield (ranging from 30% to 12%). Attempts to use an activated carbonyl group (e.g. the use of acyl chloride derivative of the esters) did not result in increased yield. The compounds synthesised were not evaluated against 5aR due to the lack of an assay system.

Estrone sulfatase (E 1 STS) belongs to a family of enzymes, namely the steroid sulfatases, which catalyse the conversion of the biologically inactive .cornpound to the more potent biologically active steroid. In particular, E1STS catalyses the conversion of estrone sulfate to estrone (E 1) and is therefore a pivotal enzyme in the progression of hormone-dependent breast cancer in postmenopausal women. The use of aromatase (AR) inhibitors, such as anastrozole, has led to the reduction of plasma levels of E1 by as much as 98%, however, it has been suggested that the high levels of E1 found within breast cancer cells are due 'to the activity of E1STS and is therefore a non-AR route which is not affected through the use of AR inhibitors. A number of compounds, both steroidal and non-steroidal, have been synthesised and subsequently evaluated as inhibitors of E1STS, however, from the ranges of compounds evaluated, 667-COUMATE remains the only compound to enter Phase I clinical trials. Within our own group, we have previously synthesised a number of compounds based on the 4-sulfamoylated derivatives of a series of alkyl 4- hydroxybenzoic acid esters, indeed, the cyclo-octyl derivative was found to be more potent than 667 -COUMATE. However, these compounds are known to be unstable in the plasma due to the presence (and action) of esterases. As such, we considered the synthesis of 4-sulfonated derivatives of both the mono- and di-substitiuted N-alkyl-4- hydroxybenzamide. In the synthesis of the target compounds, we utilised a reaction scheme which involved the initial synthesis of the mono- and di-substituted N-alkyl-4- hydroxybenzamide followed by the conversion of the 4-hydroxy moiety to the sulfamate, methansulfonate or trifluoromethansulfonate derivatives. However, a number of difficulties in the synthesis and purification of the N-alkyl-4-hydroxybenzamide from 4- hydroxybenzoic acid led us to consider the use of a protecting group for the' 4-hydroxy moiety. As such, we considered (and utilised) the initial synthesis of 4-acetoxybenzoic acid followed by the synthesis of N-alkyl-4-hydroxybenzamide via the appropriate acyl chloride. The conversion of the 4-hydroxy moiety to the sulfonate derivatives involved the reaction between N-alkyl-4-hydroxybenzamide and the appropriate sulfonyl chloride. In general, the reactions proceeded in moderate to good yield with a few problems, namely the purification of the target compounds. Although the sulfonated products are currently undergoing biochemical evaluation, initial pKa (a physicochemical factor which has been shown to play an important role in determining biochemical activity) studies suggest that the benzamide-based compounds are potentially weak inhibitors of E1STS. In an effort to produce novel inhibitors of E1STS, the synthesis of non-phenolic based inhibitors was also considered within the current study. In particular, synthesis of sulfamoylated derivatives of alkyl and benzyl alcohols was undertaken involving a reaction between the appropriate alcohol and aminosulfonyl chloride. In general, the reactions proceeded in low to good yield with the main problem once again being the purification of the product. These compounds are also currently undergoing biochemical evaluation, however, from molecular modelling studies undertaken within the group, they would initially appear to be weak inhibitors of E1STS. Within this range, however, the a-substituted halogen containing compounds (as these possess the potential to stabilise the alkoxide ion being formed as a result of the hydrolysis of the sulfamate moiety) may be the more potent inhibitors in comparison to the non-halogenated compounds.

Cellular materials are all around us. They can be found in nature as in bone, wood, leaves and even in our food. In the last fifty years, man has produced many synthetic cellular materials: firstly with polymeric foams and more recently with foamed metals, ceramics and glass. Polymer foams are used in a variety of applications ranging from coffee mugs to the feet of the Apollo Lunar Module, for which they were used as shock absorbers. This project was aimed at understanding the recovery from long-term compression of rigid polymer foams. Understanding the dynamics involved in the recovery process of foams is very important, especially in the automotive industry where it determines safety of the driver, passengers and pedestrians, for example, in car bumpers. In this study, foam samples were compressed by strains which spanned their linear elastic and stress plateau regions, i.e. 2.5% - 35% for one month at various temperatures. Recovery occurred in two stages, designated phase 1 and phase 2. Phase 1 is the initial recovery, which dominates the full recovery process and is complete within hours or days. Phase 2 is a lesser recovery occurring over a much longer period of approximately 100 days. The initial recovery is associated with the polymer itself, whilst phase 2 recovery is associated with the cellular structure. Recovery of all samples was monitored for a minimum of 100 days at ambient temperature. Tests were also carried out to see how the environmental surroundings affect the polymer recovery. The different polymer foams which were investigated were: • Polyethylene • Polyetherimide • Polyurethane • Polysulphone The polymers tested all showed very different responses to the changes in temperature. All polymers investigated at different compressive strains demonstrated reproducible Arrhenius plot slopes under different conditions and hence a reasonably reproducible set of values of recovery process. Analyses were based on the final total recovery of the thickness as the most reliable parameter of recovery. It has been demonstrated that the mechanism of polymer deformation and recovery probably does not involve chain scission but backbone vibration; that the best parameter for characterising the recovery process is the final total dimensional recovery of the sample; and that subtle environmental changes have a large effect on the recovery from compression, although temperature and humidity are not responsible.