The effects of oxygen free radicals on the carbohydrate moiety of IgG

The CH2-linked glycoform of rheumatoid IgG is abnormal in having a reduced galactose content. This has been postulated to be a synthetic defect due to a decrease in the level of rheumatoid B cell galactosyltransferase. However, more recent work has indicated that agalactosylation may be common to chronic inflammatory diseases. In this work we have investigated the effect of oxygen free radicals (OFRs), which are generated by activated phagocytic cells at inflammatory sites, on the carbohydrate moiety of IgG. Radiolytically generated peroxy (ROO.) and hydroxyl radicals (OH.) but not superoxide anion radicals (O2.-) were found to destroy galactose on IgG. After OH. attack, this was associated with an increase in the availability of N-acetylglucosamine, possibly due to its presence as a terminal residue. These results suggest that the agalactosylation associated with chronic inflammation may not only be synthetic in nature, but may also be a consequence of post-synthetic degradation by OFRs.

Combinatorial solid phase synthesis of multiply substituted 1,4-benzodiazepines and affinity studies on the CCK2 receptor (Part 1)

One hundred sixty-eight multiply substituted 1,4-benzodiazepines have been prepared by a five-step solid-phase combinatorial approach using syn-phase crowns as a solid support and a hydroxymethyl-phenoxy-acetamido linkage (Wang linker). The substituents of the 1,4-benzodiazepine scaffold have been varied in the -3, -5, -7, and 8-positions and the combinatorial library was evaluated in a cholecystokinin (CCK) radioligand binding assay. 3-Alkylated 1,4-benzodiazepines with selectivity towards the CCK-B (CCK2) receptor have been optimized on the lipophilic side chain, the ketone moiety, and the stereochemistry at the 3-position. Various novel 3-alkylated compounds were synthesized and [S]3-propyl-5-phenyl-1,4-benzodiazepin-2-one, [S]NV-A, has shown a CCK-B selective binding at about 180 nM. Fifty-eight compounds of this combinatorial library were purified by preparative TLC and 25 compounds were isolated and fully characterized by TLC, IR, APCI-MS, and 1H/13C-NMR spectroscopy.

Synthesis and evaluation of asperlicin analogues as non-peptidal cholecystokinin-antagonists

The SAR of Asperlicin analogues is reported, leading to bioactive 1,4-benzodiazepine-2-ones, which were prepared in a 3 step reaction sequence. The Asperlicin substructure was built up using Tryptophan and readily available 2-amino-acetophenones. This template, containing a 1,4-benzodiazepin-2-one moiety with a 3-indolmethyl side chain, was transformed into mono- and di-substituted 3-indol-3 '-yl-methyl-1,4-benzodi-azepine-2-ones by selective alkylation and acylation reactions. The SAR optimization of the 1,4-benzodiazepine scaffold has included variations at the 5-, 7-, 8-position, at the N1, N-indole nitrogen and the configuration of the C3-position. The most active Asperlicin analogue, having an IC50 of 1.6 microM on the CCKA receptor subtype, was obtained from Tryptophan in only 3 steps in an overall yield of 48%.

Synthesis of sulforaphane and inhibition of cytochrome P450 enzymes as a basis for antigenotoxicity

Many dietary factors have been associated with a decreased risk of developing cancer. One potential mechanism by which these factors, chemopreventors, protect against cancer may be via alteration of carcinogen metabolism. The broccoli constituent sulforaphane (1-isothiocyanate-4-methylsulinylbutane) (CH3-S0-(CH2)4-NCS) has been isolated as a potential inducer of phase II detoxification enzymes and also protects rodents against 9,10-dimethyl-1,2-benz[aJanthracene-induced mammary tumours. The ability of sulforaphane to also modulate phase I activation enzymes (cytochrome P450) (CYP450) was studied here. Sulforaphane was synthesised with an overall yield of 15%, essentially via 1-methylsulfinylphthalimidobutane, which was oxidised to the sulfoxide moiety. Deprotective removal of phthalimide yielded the amine, which was converted into sulforaphane by reaction with N,N'-thionocarbonyldiimidazole. Purity (95 %) was checked by 1H-NMR,13C-NMR and infrared and mass spectrometry.Sulforaphane was a competitive inhibitor of CYP2E1 in acetone-induced Sprague-Dawley rat microsomes (Ki 37.9 ± 4.5μM), as measured by the p-nitrophenol hydroxylase assay. Ethoxyresorufin deethylase activity (EROD), a measurement of CYP1A activity, was also inhibited by sulforaphane (100μM) but was not competitive, and a preincubation time-dependence was observed. In view of these results, the capacity of sulforaphane to inhibit N-nitrosodimethylamine (NDMA)-induced genotoxicity (CYP2E1-mediated) was studied using mouse liver activation systems. Sulforaphane (>0.8μM) inhibited the mutagenicity of NDMA (4.4 mg/plate) in Salmonella typhimurium strain TA100 after pre-incubation for 45 min with acetone-induced liver 9000 g supernatants from Balb/c mice. Unscheduled DNA synthesis induced by NDMA (33μ5 M) in mouse hepatocytes was also reduced by sulforaphane in a concentration-dependent manner (0.064-20μM). Sulforaphane was not genotoxic itself in any of these systems and cytotoxic only at high concentrations (>0.5 mM and > 40μM respectively). The ability of sulforaphane to modulate the orthologous human enzymes was studied using a human epithelial liver cell line (THLE) expressing individual human CYP450 isoenzymes. Using the Comet assay (a measurement of DNA strand breakage under alkaline conditions), NDMA (0.01-1μg/ml) and IQ (0.1-10μg/ml) were used to produce strand breaks in T5-2E1 cells (expressing human CYP2E1) and T5-1A2 cells (expressing human CYP1A2) respectively, however no response was observed in T5-neo cells (without CYP450 cDNA transfection). Sulforaphane inhibited both NDMA and IQ-induced DNA strand breakage in a concentration-dependent manner (0.1-10μM).The inhibition of metabolic activation as a basis for the antigenotoxic action of sulforaphane in these systems (bacteria, rodent hepatocytes and human cells) is further supported by the lack of this chemopreventor to influence NaN3 mutagenicity in S. typhimurium and H202-induced DNA strand breakage in T5-neo cells. These findings suggest that inhibition of CYP2E1 and CYP1A by sulforaphane may contribute to its chemoprotective potential.

Reactions of directly related tellurium and selenium heterocycic compunds with triiron dodecacarbonyl

The reactions of directly related tellurium and selenium heterocyclic compounds with triiron dodecacarbonyl are described. The reaction of 2-telluraphthalide, C8H8OTe with [Fe3(CO)12 gave [Fe{C6H4(CH2)Te}(CO)3]2, (1). An iron atom has inserted into the telluracyclic ring, and it is probable that one co-ordinated CO ligand arises from the initially organic carbonyl group. X-ray analysis of compound (1) showed that the compound has a Fe2Te2 core, which is achieved by dimerisation. The reaction of telluraphthalic anhydride, C8H402Te with [Fe3(CO)12] gave a known, but unexpected, organic phthalide product, C8H602, which was confirmed by X-ray crystallography. Selenaphthalic anhydride,  C8H4O2Se gave intractable products on reaction with [Fe3(CO)12], 2-selenaphthalide, C8H6OSe, on reaction with [Fe3(CO)12] gave a major product [Fe2{C6H4(CH2)Se}(CO)6], (2) and a minor product [Fe3{C6H4(CH2)Se}(CO)8], (3) which is an intermediate in the formation of (2). X-ray analysis of (2) shows that compound (2) is very similar to (1) except that the 18 electron rule is satisfied by co-ordination of a Fe(CO)3 moiety, rather than dimerisation. Compound (3), also studied by X-ray crystallography, differs from (2) mainly in the addition of an Fe(CO)2 moiety. Telluraphtbalic anhydride, C8H402Te, and selenaphthalic anhydride, C8H402Se, are both monoclinic and crystallise in space group P21/n. 2-Selenaphthalide, C8H402Se, is also monoclinic, space group P21/C. The reactions of the following compounds (l,3-dihydrobenzo[c]selenophene, 1,3,7,9-tetrahydrobenzo[1,2c; 4,5c'] ditellurophene, dibenzoselenophene, phenoxselenine, 3, 5-naphtho-1-telluracyclohexane and 3,5-naphtho-1-selenacyclohexane) with [Fe3lCO)12] are reported. It is unfortunate that the above compounds do not react under the conditions employed; this may be due to differing degrees of ring strain. 1,8-bis(bromomethyl)naphthalene, C12H10Br2 is monoclinic and crystallises in space group C2/c. 1,1-diiodo-3,5-naphthotelluracyclohexane, C12H10TeI2 and 3,5-naphtho-l-telluracyclohexane, C12H10Te are monoclinic and crystallise in space group P21/c. 3,5-naphtho-l-selenacyclohexane, C12H10Se and 2,2,8,8-tetraiodo-1,3,7,9-tetrahydrobenzo[1,2c;4,5c']ditellurophene are also monoclinic, space group P21/a. The syntheses of intramolecular stabilised organo-tellurium and selenium compounds are reported, having a general formula of REX (where R = phenylazophenyl; E = Se, Te; X = electronegative group, for example C1, Br or I). The crystal structures of R'TeBr, RTeI, RSeCI, RSeCI/I and RSeI (where R = phenylazophenyl) are reported. The tellurium containing X-ray structures are triclinic and have a space group P-1. The selenium containing X-ray structures are monoclinic with space group P21/n. The inclusion of nitrogen in selenium heterocycles provides access to an entirely new area of organometallic chemistry. The reaction of 2-methylbenzoselenazole with [Fe3(CO)12] gave [Fe2{C6H4(NCH2CH3)Se}(CO)6]. The reactions of 2-(methyltelluro)benzanilide or 2-(methylseleno)benzanilide with [Fe3(CO)12] gave reaction products [Fe2(μTeMe)2(CO)6] and [Fe2 (μ-SeMe)2(CO)6] respectively, which were confmned by X-ray crystallography. The use of Mossbauer spectroscopy on the products obtained from the reactions of heterocyclic compounds with [Fe3(CO)12] can give useful information, for example the number of iron sites and the environments of these iron sites within the products.

Migration of additives from thermoplastic polymers

A homologous series of ultra-violet stabilisers containing 2-hydroxybenzophenone (HBP) moiety as a uv absorbing chromophore with varying alkyl chain lengths and sizes were prepared by known chemical synthesis. The strong absorbance of the HBP chromophore was utilized to evaluate the concentration of these stabilisers in low density polyethylene films and concentration of these stabilisers in low density polyethylene films and in relevant solvents by ultra-violet/visible spectroscopy. Intrinsic diffusion coefficients, equilibrium solubilities, volatilities from LDPE films and volatility of pure stabilisers were studied over a temperature range of 5-100oC. The effects of structure, molecular weight and temperature on the above parameters were investigated and the results were analysed on the basis of theoretical models published in the literature. It has been found that an increase in alkyl chain lengths does not change the diffusion coefficients to a significant level, while attachment of polar or branched alkyl groups change their value considerably. An Arrhenius type of relationship for the temperature dependence of diffusion coefficients seems to be valid only for a narrow temperature range, and therefore extrapolation of data from one temperature to another leads to a considerable error. The evidence showed that increase in additive solubility in the polymer is favoured by lower heat of fusions and melting points of additives. This implies the validity of simple regular solution theory to provide an adequate basis for understanding the solubility of additives in polymers The volubility of stabilisers from low density polyethylene films showed that of an additive from a polymer can be expressed in terms of a first-order kinetic equation. In addition the rate of loss of stabilisers was discussed in relation to its diffusion, solubility and volatility and found that all these factors may contribute to the additive loss, although one may be a rate determining factor. Stabiliser migration from LDPE into various solvents and food simulants was studied at temperatures 5, 23, 40 and 70oC; from the plots of rate of migration versus square root time, characteristic diffusion coefficients were obtained by using the solution of Fick's diffusion equations. It was shown that the rate of migration depends primarily on partition coefficients between solvent and the polymer of the additive and also on the swelling action of the contracting media. Characteristic diffusion coefficients were found to approach to intrinsic values in non swelling solvents, whereas in the case of highly swollen polymer samples, the former may be orders of magnitude greater than the latter.

Cyclodextrin complexes of antimicrobial agents

Poorly water-soluble drugs show an increase in solubility in the presence of cyclodextrins (CyD) due to the formation of a water-soluble complex between the drug and dissolved CyD. This study investigated the interactions of -Cyd and hydroxypropyl--CyD (HP--CyD, M.S. = 0.6) with antimicrobial agents of limited solubility in an attempt to increase their microbiological efficacy. The agents studied were chlorhexidine dihydrochloride (CHX), p-hydroxybenzoic acid esters (methyl, ethyl, proply and butyl) and triclosan. The interactions between the antimicrobials and CyDs were studied in solution and solid phases. Phase solubility studied revealed an enhancement in the aqueous drug solubility in the presence of the CyD and also gave an indication of the complex stability constant (Ks). The temperature-dependence of the stability constant of the complex was modelled by the van't Hoff plot which yielded the thermodynamic parameters for complexation. Further confirmation of the inclusion of the antimicrobials within the cavity of the CyDs in aqueous solution was obtained from proton magnetic resonance and ultraviolet absorption spectroscopies. The former method indicated that the chlorophenyl moiety of the CHX was included within the -CyD cavity and the stoichiometry of the complex formed was 1:1. The solid-phase complexes were prepared by freeze-drying. The inclusion complex of triclosan with HP--CyD was obtained from aqueous solution with the addition of ammonia. Evidence to confirm complex formation was obtained from DSC, IR and X-ray powder diffraction studies. Dissolution studies of the solid inclusion complexes using the dispersed powder technique illustrated their superior solubilities as compared to the equimolar physical mix of the guest and CyD. It was shown that these solutions of the complex were supersaturated with respect to the free guest. This was further demonstrated by diffusion studies which showed the flux of free drug from donor solutions of the antimicrobial-CyD complex to be significantly greater than the flux from donor suspensions of drug alone.

Combinatorial approach to multi-substituted 1,4-Benzodiazepines as novel non-peptide CCK-antagonists

For the drug discovery process, a library of 168 multisubstituted 1,4-benzodiazepines were prepared by a 5-step solid phase combinatorial approach. Substituents were varied in the 3,5, 7 and 8-position on the benzodiazepine scaffold. The combinatorial library was evaluated in a CCK radiolabelled binding assay and CCKA (alimentary) and CCKB (brain) selective lead structures were discovered. The template of CCKA selective 1,4-benzodiazepin-2-ones bearing the tryptophan moiety was chemically modified by selective alkylation and acylation reactions. These studies provided a series of Asperlicin naturally analogues. The fully optimised Asperlicin related compound possessed a similar CCKA activity as the natural occuring compound. 3-Alkylated 1,4-benzodiazepines with selectivity towards the CCKB receptor subtype were optimised on A) the lipophilic side chain and B) the 2-aminophenyl-ketone moiety, together with some stereochemical changes. A C3 unit in the 3-position of 1,4-benzodiazepines possessed a CCKB activity within the nanomolar range. Further SAR optimisation on the N1-position by selective alkylation resulted in an improved CCKB binding with potentially decreased activity on the GABAA/benzodiazepine receptor complex. The in vivo studies revealed two N1-alkylated compounds containing unsaturated alkyl groups with anxiolytic properties. Alternative chemical approaches have been developed, including a route that is suitable for scale up of the desired target molecule in order to provide sufficient quantities for further in vivo evaluation.

The synthesis and evaluation of small organic molecules as cholecystokinin antagonists

Cholecystokinin (CCK) is a peptide hormone, present in the alimentary and the CNS. It is the most abundant peptide in the brain. CCK has been implicated in a number of disorders. The link between CCK and anxiety was the basis for this research. A comprehensive discussion on the many types of CCK receptor antagonists is included. For the drug discovery process, a number of synthetic approaches have been investigated and alternative chemical approaches developed. 1,4-Benzodiazepine analogues were prepared, with substitutents In the 1,2 & 3- position of the benzodiazepine scaffold varied, and substituted 3-anilino benzodiazepines exhibited the greatest in vitro activity towards the CCKA receptor subtype. Through extensive screening, pyrazolinone-ureido derivatives were identified, optimised, SAR studied and re-screened. A comprehensive in vivo study on the most active analogue is included, which has a number of common structural features with L-36S, 260 including activity. Pyrazolinone-amide derivatives, bearing the tryptophan moiety were equally active. A number of existing and novel furan- 2(SH)-one building blocks were prepared, from which a selected mini-library of 4- amino-substituted furan-2(SH)-ones were prepared and evaluated. All synthesised compounds were evaluated in a CCK radiolabelled binding assay (CCKA & CCKB), with compounds demonstrating receptor selectivity and lead structures being discovered. The work in this thesis has identified a number of highly active prime structures, from which further investigations are essential in providing more in vitro & in vivo data and the need to prepare more analogues.

Design, synthesis and evaluation of cyclothialidine analogues as DNA gyrase inhibitors

Cyclothialidine, a natural product isolated from Streptomyces .filipinensis NR0484, has been proven to be a potent and selective inhibitor of the bacterial enzyme DNA gyrase. Gyrase inhibition results in cell death, the enzyme being the target of several currently used antibiotics. Cyclothialidine showed poor activity against whole bacterial cells, highlighting scope for improvement regarding cell membrane pemeability in order for the full potential of this new class of antibiotics to be realised, Structurally, cyclothialidine contains a 12-membered lactone ring which is partly integrated into a pentapeptide chain, with a substituted aromatic moiety bordering the lactone, Retrosynthetically it can be traced back to cis-3-hydroxyproline, 3,5-dihydroxy-2,6-dimethylbenzoic acid and four commercially available amino acids; two serine, one cysteine and one alanine. In this work, a model of cyclothialidine was synthesised in order to establish the methodology for more complex compounds. Analogues with hydroxy, dihydroxy and dihydroxymethyl substituted aromatic moieties were then prepared to ensure successful protection methods could be performed and the pharmacophore synthesised. The key aromatic moiety, 2,6-dimethyl-3,5-dihydroxybenzoic acid was produced via two successive Mannich reaction/reduction steps. Acid protection using 4-nitrobenzyl bromide and TBDMS hydroxyl protection followed by bromination of one methyl afforded the desired intermediate. Reaction with a serine/cysteine dipeptide, followed by deprotection and cyclisation under Mitsunobu conditions lead to the 12-membered lactone. An amine substituted aromatic analogue and also replacement of the cysteine sulphur by oxygen were attempted but without success. In an effort to improve cell permeability, a conjugate was synthesised between the pharmacophore and a cholesterol moiety. It was hoped the steroid fragment would serve to increase potency by escorting the molecule through the lipid environment of the cell membrane. The pharmacophore and conjugate were tested against a variety of bacterial strains but the conjugate failed to improve activity.

The metabolism of n-methyl compounds

The metabolism of compounds containing the N-methyl group is discussed with particular consideration being made to the possible role of the product of oxidative metabolism, the N-hydroxymethyl moiety, in the generation of potentially toxic, reactive electrophiles. Particular pathways which are considered are: (i), the production of formaldehyde; (ii), the generation of iminium ions or imines; and (iii), the formation of N-formyl compounds which might act as formylating agents. 4-Chloro-N-(hydroxymethyl)benzamide and 3-(4-chlorophenyl)-1-hydroxy-methyl-1-methylurea (the product of oxidative metabolism of 3-(4-chlorophenyl)-1,1-dimethylurea) are model carbinolamides which do not readily release formaldehyde. The electrophilic properties of these model carbinolamides were investigated: neither reacted with nucleophiles such as cyanide or glutathione under physiological conditions. In contrast, N-(acetoxymethyl)-4-chlorobenzamide yielded the cyanomethylamide with potassium cyanide and S-(4-chlorobenzamidomethyl)glutathione with glutathione. 4-Chloro-N-(hydroxymethyl)benzamide and 3-(4-chlorophenyl)-1,1-dimethylurea were not biotransformed to electrophilic moieties when incubated with mouse hepatic 9000 x g supernatant and Acetyl-CoA or PAPS-generating system. N-(Acetoxymethyl)-4-chlorobenzamide was non-mutagenic to Salmonella typhimurium in the short term bacterial assay; but toxicity to the bacteria was observed. 4-Chloro-N-(hydroxymethyl)benzamide and 3-(4-chlorophenyl)-1,1-dimethylurea showed no mutagenicity or toxicity in the mutagenicity assay including an Aroclor-induced rat hepatic 9000 x g supernatant. Addition of Acetyl-CoA or a PAPS-generating system did not produce a mutagenic response. 4-Chloro-N-formlbenzamide did not act as a formylating agent towards the weak nucleophile aniline. However, 4-chloro-N-formylbenzamide, N-formylbenzamide, 3-(4-chlorophenyl)-1-formyl-1-methylurea and 3-(4-chlorophenyl)-1-formylurea are all metabolised by mouse hepatic mirosomes and post-microsomal supernatant. The results demonstrate the potential for N-hydroxymethyl compounds to generate highly reactive species if these are substrates for conjugation with sulphate (or acetate). The model compounds employed here, apparently do not show any ability to be conjugated themselves, however, other N-hydroxymethyl compounds might be readily conjugated. The formation of N-formyl compounds does not appear to be toxicologically significant, as adjudged on limited experiments performed, but rather represent a detoxification pathway.

Studies of the metabolism and the toxicity of N-Methylformamide and related amides

The hepatotoxicity of the industrial solvent and investigational anti-tumour agent N-methylformamide (NMF, HOCNHCH3) and several structural analogues was assessed in mice. NMF and its ethyl analogue (NEF) were equipotent hepatotoxins causing extensive centrilobular necrosis and damage to the gall bladder. Pretreatment of mice with SKF525A did not influence the toxicity of these N-alkylformamides. Replacement of the formyl hydrogen of NMF with deuterium or methyl significantly reduced its hepatotoxicity. An in vitro model for the study of the toxicity and metabolism of N-alkylformamides was developed using isolated mouse hepatocytes. The cytotoxicity of NMF in vitro was concentration-dependent with maximal toxicity being achieved at concentrations of 5mM or above. The cytotoxic potential of related amides correlated well with their in vivo hepatotoxic potential. Pretreatment of mice with buthionine sulphoximine (BSO), which depleted hepatocytic levels of glutathione to 15% of control values, exacerbated the cytotoxicity of NMF towards the hepatocytes. NMF (1mM or above), incubated with isolated mouse hepatocytes, depleted intracellular glutathione levels to 26% of control values within 4h. Depletion of glutathione was quantitatively matched by the formation of a carbamoylating metabolite. Metabolism was dependent on the concentration of NMF and was drastically reduced in incubations of hepatocytes isolated from mice pretreated with BSO. The carbamoylating metabolite, S-(N-methylcarbamoyl)-glutathione (SMG), was identified in vitro using FAB-MS. The generation of SMG was subject to a large primary H/D kinetic isotope effect when the formyl hydrogen was replaced with deuterium. Likewise, glutathione depletion and metabolite formation were reduced or abolished by the deuteration or methylation of the formyl moiety of NMF. NEF, like NMF, depleted hepatocytic glutathione levels and was metabolised to a carbamoylating metabolite. Radioactivity derived from 14C-NMF and 14C-NEF, labelled in the alkyl moieties, was found to be irreversibly associated with microsomal protein on incubation in vitro. Binding was dependent on the presence of NADPH and was mostly abolished in the presence of reduced glutathione. SKF525A failed to influence the binding.

The design of novel antifolates against pneumocystis carinii

The pneumonia caused by Pneumocystis carinii is ultimately responsible for the death of many acquired immunodeficiency syndrome (AIDS) patients. Large doses of trimethoprim and pyrimethamine in combination with a sulphonamide and/or pentamidine suppress the infection but produce serious side-effects and seldom prevent recurrence after treatment withdrawal. However, the partial success of the aforementioned antifolates, and also trimetrexate used alone, does suggest dihydrofolate reductase (DHFR) as a target for the development of antipneumocystis agents. From the DHFR inhibitory activities of 3'-substituted pyrimethamine analogues it was suggested that the 3'-(3'',3''-dimethyltriazen-1''-yl) substituent may be responsible for the greater activity for the P.carinii over the mammalian enzyme. Crystallographic and molecular modeling studies revealed considerable geometrical and electronic differences between the triazene and the chemically related formamidine functions that may account for the differences in DHFR inhibitory profiles. Structural and electronic parameters calculated for a series of 3'-(3'',3''-disubstitutedtriazen-1''-yl) pyrimethamine analogues did not correlate with the DHFR inhibitory activities. However, the in vitro screening against P.carinii DHFR revealed that the 3''-hydroxyethyl-3''-benzyl analogue was the most active and selective. Models of the active sites of human and P.carinii DHFRs were constructed using DHFR sequence and structural homology data which had identified key residues involved in substrate and cofactor binding. Low energy conformations of the 3'',3''-dimethyl and 3''-hydroxyethyl-3''-benzyle analogues, determined from nuclear magnetic resonance studies and theoretical calculations, were docked by superimposing the diaminopyrimidine fragment onto a previously docked pyrimethamine analogue. Enzyme kinetic data supported the 3''-hydroxyethyl-3''-benzyl moiety being located in the NADPH binding groove. The 3''-benzyl substituent was able to locate to within 3 AA of a valine residue in the active site of P.carinii DHFR thereby producing a hydrophobic contact. The equivalent residue in human DHFR is threonine, more hydrophilic and less likely to be involved in such a contact. This difference may account for the greater inhibitory activity this analogue has for P.carinii DHFR and provide a basis for future drug design. From an in vivo model of PCP in immunosuppressed rats it was established that the 3"-hydroxyethyl-3"-benzyl analogue was able to reduce the.P.carinii burden more effectively with increasing doses, without causmg any visible signs of toxicity. However, equivalent doses were not as effective as pentamidine, a current treatment of choice for Pneumocystis carinii pneumonia.

Non-classical inhibitors of dihydrofolate reductase

This thesis comprises two main objectives. The first objective involved the stereochemical studies of chiral 4,6-diamino-1-aryl-1,2-dihydro-s-triazines and an investigation on how the different conformations of these stereoisomers may affect their binding affinity to the enzyme dihydrofolate reductase (DHFR). The ortho-substituted 1-aryl-1,2-dihydro-s-triazines were synthesised by the three component method. An ortho-substitution at the C6' position was observed when meta-azidocycloguanil was decomposed in acid. The ortho-substituent restricts free rotation and this gives rise to atropisomerism. Ortho-substituted 4,6-diamino-1-aryl-2-ethyl-1,2-dihydro-2-methyl-s-triazine contains two elements of chirality and therefore exists as four stereoisomers: (S,aR), (R,aS), (R,aR) and (S,aS). The energy barriers to rotation of these compounds were calculated by a semi-empirical molecular orbital program called MOPAC and they were found to be in excess of 23 kcal/mol. The diastereoisomers were resolved and enriched by C18 reversed phase h.p.l.c. Nuclear overhauser effect experiments revealed that (S,aR) and (R,aS) were the more stable pair of stereoisomers and therefore existed as the major component. The minor diastereoisomers showed greater binding affinity for the rat liver DHFR in in vitro assay. The second objective entailed the investigation into the possibility of retaining DHFR inhibitory activity by replacing the classical diamino heterocyclic moiety with an amidinyl group. 4-Benzylamino-3-nitro-N,N-dimethyl-phenylamidine was synthesised in two steps. One of the two phenylamidines indicated weak inhibition against the rat liver DHFR. This weak activity may be due to the failure of the inhibitor molecule to form strong hydrogen bonds with residue Glu-30 at the active site of the enzyme.

Organic synthesis and fungicidal activity of oxylipin-based compounds

Previous research has shown that the naturally occurring reactive electrophilic species (RES), 12-oxophytodienoic acid (OPDA), not only serves as a precursor for jasmonic acid but is also a potent antifungal compound. However, both the low amount present in plants and the multistep synthesis required to produce this compound on a scale viable for agrochemical use currently limits its practical value. The aim of this research was to generate a range of molecular mimics of OPDA with a minimum number of synthetic steps and screen for antifungal activity. Synthetic 4-octyl-cyclopentenone containing the cyclopentenone ring and an eight carbon alkyl chain was found to show the highest in vitro antifungal activity against C. herbarum and B. cinerea with minimum inhibition concentration (MIC) of 100-200µM. This indicates that structurally simplified 4-octyl-cyclopentenone can be successfully synthesised to mimic the antifungal activity of OPDA against specific fungal strains. Application of 4-octyl-cyclopentenone could act as surfactant by disrupting and disorganising the lipid membrane non-specifically, resulting in the leakage of potassium ions, which was the proposed mode of action of this compound. However, the sensitivity of fungi to this compound is not correlated to the lipid composition of fungal spores. (E)-2-alkenals were also studied for their antimicrobial activity and (E)-2-undecenal was found to have the highest antimicrobial activity against a range of pathogens. The hydrophilic moiety (the a,ß-unsaturated carbonyl group), common to both (E)-2-undecenal and 4-octyl-cyclentenone is essential to their bioactivity, and the hydrophobic moiety plays an important role in their antimicrobial activities. 4-Octyl-cyclopentenone showed no visible toxicity to the test plant, Arabidopsis thaliana, suggesting that its high antifungal activity against Botrytis and Cladosporium could be exploited for commercialisation as a new generation of agrochemical.