In this thesis the production, isolation and purification of the kalimantacin antibiotics are described along with details of the elucidation of the structures including the relative and absolute stereochemistry. Kalimantacin A, isolated from Pseudomonas jluorescens strain BCCM_ ID9359, is biosynthesised by a trans-AT type I polyketide synthase (PKS) and exhibits strong and selective activity against multi-resistant Staphylococcus aureus (MRSA). Two mutant strains, ~batF and ~batM, have been produced from which 17- hydroxy analogue 20, 27-descarbamoyl analogue 21 and 17-hydroxy-27-descarbamoyl analogue 22 were isolated and fully characterised.

This thesis is concerned with the use of vinylsulfonium salts as efficent annulation reagents for the synthesis of heterocycles. Firstly, a novel, efficient and versatile route for the formation of cyclopropane-fused hetereocycles from easily available starting materials has been developed, giving access to a range of functionalised 3-azabicyclo[3.1.0]hexanes in good yields and with high levels of diastereoselectivity. Treatment of easily prepared allylic amines, or aza-Morita-Baylis-Hillman adducts, with bromoethylsulfonium triflate, gave access to 3-azabicyclo[3.1.0]hexanes with both α,β-substitution (45 -63%, >20:1 dr) and a,y-substitution patterns (43-76%, up to >20:1 dr), In comparison to previous methods, this protocol enables the synthesis of a more diverse range of substituted and functionalised [3.1.0] scaffolds with very high diastereoselectivity. There is considerable interest in exploring this class of bioactive compounds, which should now be enabled by the methodology described in this work. The synthesis of a new class of vinylsulfonium salts bearing α-substituents, in three steps from commercially available styrenes, has also been achieved. An investigation into the annulation properties of this new class of reagents has revealed similar properties to the original bromoethylsulfonium triflate. Application of these a-substituted vinylsulfonium salts in the epoxyannulation of the easily prepared p-amino ketones, gave a range of 6-oxa-3-azabicyclo[3 .1.0]hexanes in moderate to good yields (41-83%) and with high levels of diastereocontrol (>20: 1 dr). Cyclopropanation of allylic ammes with a-substituted vinylsulfonium salts also gave 3-azabicyclo[3 .1.0]hexanes in moderate to good yields (45 -72%) and with high levels of diastereocontrol (>20: 1 dr). It is envisaged that this new class of reagents will further expand the scope of vinylsulfonium salt methodology, leading to further applications in both pharmaceutical and academic research.

No description available.

No description available.

No description available.

The action of thrombin is central to the processes of thrombosis and haemostasis. Thrombin is generated following the activation of prothrombin by 'prothrombinase' (II-ase), the phospholipid membrane-bound factor Xa (fXa)-factor Va (fVa) complex, which in turn is dependent upon the generation of fXa by 'intrinsic tenase' (X-ase), the phospholipid membrane-bound factor IXa (fIXa)-factor VIIIa (fVIIIa) complex. Thrombin not only converts fibrinogen to fibrin, but also amplifies its own formation by activating the cofactors in II-ase and X-ase, factor V and factor VIII, respectively. The critical role played by II-ase and X-ase towards thrombin generation makes these membrane-bound enzyme complexes attractive targets for inhibition and offers an effective approach to blocking coagulation. Heparin acts as an anticoagulant by activating antithrombin (AT), which then inactivates thrombin, fXa and other activated clotting factors. However, the heparin-AT complex has limited activity against membrane-bound fIXa and fXa. Recently, in buffer systems, heparin has been shown to have an AT-independent effect on coagulation by directly inhibiting X-ase, an effect that is minimal in plasma where the AT-dependent heparin effect predominates. To capitalise on this AT-independent effect, heparin was chemically modified by periodate oxidation and borohydrate reduction to lower its affinity for AT (from Kd value of 25nM to 43μM); we used LMWH rather than heparin to take advantage of the superior pharmacokinetic profile of LMWH. Using this low affinity LMWH (LA-LMWH), N-desulphated LMWH was prepared using a solvolytic desulphation method. Whereas LA-LMWH inhibited X-ase to a similar extent as LMWH with high AT-affinity (IC<SUB>50</SUB> of 16μg/ml and 13μg/ml respectively), N-desulphated LMWH had minimal inhibitory activity (IC<SUB>50</SUB> of 166μg/ml).

The purpose of these studies was to determine the effects and mechanisms of action of glucocorticoids on eosinophil and neutrophil apoptosis and to examine the effects of these drugs on the phagocytosis of apoptotic granulocytes by macrophages. Dexamethasone exerts diametrically opposed effects on these two cell types; promoting eosinophil apoptosis and inhibiting neutrophil apoptosis. Similarly, elevation of [Ca<SUP>2+</SUP>]<SUB>i</SUB> also differentially affects the rate of constitutive granulocyte apoptosis. In contrast, elevation of cAMP inhibits the rate of apoptosis in both granulocytes, whereas inhibition of PKC promotes the rate of granulocyte apoptosis. Moreover, inhibition of protein phosphatases inhibits the rate of granulocyte apoptosis at lower concentrations and promotes the rate of granulocyte apoptosis at higher concentrations. Inhibition of the MAP/ERK kinase cascade inhibits the rate of constitutive eosinophil apoptosis, while having no effect upon the rate of constitutive neutrophil apoptosis. However, inhibition of this cascade selectively blocks the neutrophil survival-promoting effects of LPS, but exerts no effect on the glucocorticoid-mediated neutrophil survival-promoting effects of LPS, but exerts no effect on glucocorticoid-mediated inhibition of neutrophil apoptosis. Dexamethasone mediates inhibition of neutrophil apoptosis by a PKA-dependent mechanism, whereas the pro-apoptotic effect of dexamethasone upon eosinophil apoptosis appears to the PKA-independent. Dexamethasone potentiates macrophage recognition and phagocytosis of apoptotic eosinophils and neutrophils. Demonstration of the marked apoptosis-promoting effects of corticosteroids on eosinophil apoptosis together with the augmentation of macrophage clearance of apoptotic eosinophils, may in part explain the known beneficial therapeutic effects of corticosteroids on established 'eosinophilic' inflammatory diseases such as asthma. The opposite effect on neutrophil apoptosis may underlie the lower efficacy of these drugs in 'neutrophilic' inflammatory diseases such as SRDs. Further dissection of the intracellular mechanisms governing the divergent effects of corticosteroids on eosinophil and neutrophil apoptosis may lead to new therapeutic targets with which selective induction of apoptosis could be achieved.

The actions of protein kinase C (PKC) on several aspects of cellular control of Ca<SUP>2</SUP>+ movement were investigated in rat anterior pituitary cells. Depolarising concentrations of K<SUP></SUP>+ induced influx of <SUP>45</SUP>Ca<SUP>2</SUP>+ into rat anterior pituitary prisms and into cells of the GH<SUB>3</SUB> rat anterior pituitary cell line. These responses were used as models to investigate the effects of activators and inhibitors of PKC. Depolarisation-induced <SUP>45</SUP>Ca<SUP>2</SUP>+ influx into anterior pituitary prisms and GH<SUB>3</SUB> cells was inhibited by the 'L'-type Ca<SUP>2</SUP>&43 channel blocker, nimodipine with equal potency in both tissues; suggesting that similar 'L'-type Ca<SUP>2</SUP>+ channels were being utilised in both preparations. Activators of PKC such as phorbol 12,13-dibutyrate (PDBu) and 4β-phorbol 12,13-didecanoate (4β-PDD) enhanced K<SUP></SUP>+ -induced <SUP>45</SUP>Ca<SUP>2</SUP>+ influx in anterior pituitary pieces, but inhibited K<SUP></SUP>+ -induced <SUP>45</SUP>Ca<SUP>2</SUP>+ influx into GH<SUB>3</SUB> cells. The modulation seen with these phorbol esters was stereo-specific and concentration-dependent and of a similar time course in both tissues. The phorboid, mezerein, and some related phorbol esters could mimic PDBu at enhancing K<SUP></SUP>+ -induced <SUP>45</SUP>Ca<SUP>2</SUP>+ influx into anterior pituitary pieces, whereas the same compounds did not mimic the action of PDBu in GH<SUB>3</SUB> cells, but instead enhanced K<SUP></SUP>+ -induced <SUP>45</SUP>Ca<SUP>2</SUP>+ influx into GH<SUB>3</SUB> cells. Furthermore, the PDBu-induced enhancement of K<SUP></SUP>+ -evoked <SUP>45</SUP>Ca<SUP>2</SUP>+ influx into anterior pituitary pieces and the PDBu-induced inhibition of K<SUP></SUP>+ -evoked <SUP>45</SUP>Ca<SUP>2</SUP>+ influx into GH<SUB>3</SUB> cells was reversed by the PKC inhibitors, staurosporine and H7, but not their less active congeners K252a and HA1004 respectively.

In this thesis I review the biology of endothelin as well as discuss the evidence supporting a pathophysiological role for endothelin in a number of cardiovascular and renal diseases. In the studies described, I have further investigated the physiology of the endothelin system as well as examined the pharmacology of endothelin receptor antagonists in man. Finally, I have also examined the role of endothelin in the pathophysiology of chronic renal failure and essential hypertension. Study 1: Inhibitors of the enzyme, neutral endopeptidase cause forearm vasoconstriction when infused into the brachial artery. This enzyme appears to significantly contribute to the breakdown of endothelin in vivo. Study 2: Big ET-3 is converted to the mature peptide ET-3 in the forearm circulation, but not in capacitance vessels. This study suggests the existence of a third endothelin-converting enzyme capable of converting big ET-3. Study 3: Systemic doses of TAK-044, a non-selective endothelin receptor antagonist, lowers vascular resistance and blood pressure in man, demonstrating that endothelin contributes to the maintenance of blood pressure in health. Study 4: Systemic TAK-044 completely blocks the local vasoconstriction produced by intrabrachial artery infusion of ET-1 for up to 3 hours. This finding has implications for the treatment of acute vasospastic conditions. Study 5: Systemic doses of TAK-044 only partially block ET-1 mediated vasoconstriction for 12 hours. This has important implications for dosing schedules in potential therapeutic interventions. Study 6: TAK-044 causes renal vasodilatation and lowers effective filtration fraction by a relative decrease in glomerular filtration rate and increase in effective plasma flow in healthy volunteers. This study demonstrates that endothelin has a role in controlling the renal circulation. Study 7: TAK-044 lowers systemic vascular resistance and blood pressure in patients with chronic renal failure, with a reduction in effective filtration fraction.

A number of cellular responses mediated by protein kinase C (PKC) in the anterior pituitary gland involve a form of PKC that is unusually resistant to the PKC inhibitor H7. In this study, inhibition by staurosporine, H7 and Ro 31-8220 of phorbol 12,13-dibutyrate-induced PKC activity in cytosol partially-purified from rat midbrain, anterior pituitary and a number of other tissues, as well as COS 7 cells, was studied. An <I>in vitro</I> mixed micelle histone IIIS phosphorylation assay was used to allow comparison of Ca<SUP>2+</SUP>-dependent and Ca<SUP>2+</SUP>-independent PKC activity. In anterior pituitary but not midbrain or COS 7 cells, Ca<SUP>2+</SUP>-independent activity was notably resistant to H7 but sensitive to staurosporine and Ro 31-8220. All Ca<SUP>2+</SUP>-dependent activity was sensitive to these three inhibitors. Comparison of PKC activity from a variety of different tissue sources showed H7-resistant, Ca<SUP>2+</SUP>-independent activity also occurred in lung but not in spleen, thalamus or cerebellum. Mezerein and 1,2-dioctanoyl-<I>sn</I>-glycerol also activated this H7-insensitive PKC from anterior pituitary. Phosphorylation of a number of substrates by anterior pituitary PKCs were also compared. When [Ala<SUP>9,10</SUP>,Lys<SUP>11,12</SUP>] glycogen synthase<SUB>1-12</SUB> (GS peptide) but not [Ser]<SUP>25</SUP> PKC a<SUB>19-31</SUB> was the phosphate acceptor, a clear component of the activity, which was Ca<SUP>2+</SUP>-independent, was resistant to H7. The tissue distribution of this activity and its characteristic resistance to H7 but not other inhibitors, does not obviously correlate with that of any of the well-characterised PKCs, and may reflect either a novel or a modified isoform. Analysis of clones using selected restriction endonucleases and subsequent DNA sequencing analysis identified sequences encoding PKC α, and ζ but not novel PKC related sequences.