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.

Synthesis and evaluation of antibacterial activity of the dual-action agents: beta-lactamase inhibitors with cytotoxic agents or beta-lactam antibiotics

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Comparison and characterisation of ribozyme delivery in cultured glial and neuronal cells in vivo and in vitro

Ribozymes are short strands of RNA that possess a huge potential as biological tools for studying gene expression and as therapeutic agents to down-regulate undesirable gene expression. Successful application of ribozymes requires delivery to the target site in sufficient amounts for an adequate duration. However, due to their large size and polyanionic character ribozymes are not amenable to transport across biological membranes. In this study a chemically modified ribozyme with enhanced biological stability, targeted against the EGFR mRNA has been evaluated for cellular delivery to cultured glial and neuronal cells with a view to developing treatments for brain tumours. Cellular delivery of free ribozyme was characterised in cultured glial and neuronal cells from the human and rat. Delivery was very limited and time dependent with no consistent difference observed between glial and neuronal cells in both species. Cellular association was largely temperature and energy-dependent with a small component of non-energy dependent association. Further studies showed that ribozyme cellular association was inhibited with self and cross competition with nucleic and non-nucleic acid polyanions indicating the presence of cell surface ribozyme-binding molecules. Trypsin washing experiments further implied that the ribozyme binding surface molecules were protein by nature. Dependence of cellular association on pH indicated that interaction of ribozyme with cell surface molecules was based on ionic interactions. Fluoresence studies indicated that, post cell association, ribozymes were sequestered in sub-cellular vesicles. South-Western blots identified several cell surface proteins which bind to ribozymes and could facilitate cellular association. The limited cellular association observed with free ribozyme required the development and evaluation of polylactide-co-glycolide microspheres incorporating ribozyme for enhanced cellular delivery. Characterisation of microsphere mediated delivery of ribozyme in cultured glial and neuronal cells showed that association increased by 18 to 27-fold in all cell types with no differences observed between cell lines and species. Microsphere mediated delivery was temperature and energy dependent and independent of pH. In order to assess the potential of PLGA micro spheres for the CNS delivery of ribozyme the distribution of ribozyme entrapping microspheres was investigated in rat CNS after intracerebroventricular injection. Distribution studies demonstrated that after 24 hours there was no free ribozyme present in the brain parenchyma, however microsphere entrapped ribozyme was found in the CNS. Microspheres remained in the ventricular system after deposition and passed from the lateral ventricles to the third and fourth ventricle and in the subarachnoid space. Investigation of the influence of microsphere size on the distribution in CNS demonstrated that particles up to 2.5 and O.5f.lm remained in the ventricles around the choroid plexus and ependymal lining.

Synthesis and characterization of triazolotriazines

The Dimroth rearrangement in ring-fused 1,2,4-triazoles has been reviewed in detail in Part I and the synthesis of all known triazolo-triazines is described in Part II. Experimental investigations concerned the establishinent of the skeletal arrangement of a variety of triazolotriazines formed by several synthetic routes. Interaction of 3-amino-5-hydrazino-12,4-triazole and benzilafforded 2-amino-6, 7-diphenyl-1, 2,4-triazolo[ 5, 1-c-]-1,2,4-triazine,whereas cyclization of 5,6-diphenyl-3-hydrazino-1,2,4-triazine withcyanogen bromide resulted in the isomeric 3-amino-6,7-diphenyl,-1,2,4-triazolo [4, 3-b]-1,2,4-triazine: both amines were deaminated with amyl nitrite in boiling tetrahydrofuran without rearrangement of the heterocyclic skeleton. 6,7-Diphenyl-1,2,4-triazolo[5,1-cJ-1,2.4-triazine, synthesized from 3-hydrazino-1,2,4-triazole and benzil, formed a covalent hydrate which could be detected spectroscopically in solution, and a covalemt methanolate and ethanolate which could be isolated. A new route to 3-amino-5-hydrazino-pyrazole is described and cyclization to 7-amino-3,4-diphenyl-pyrazolo[ 5,1-.c]-1,2,4-triazine was achieved with benzil. The diazonium nitrate of 3-amino-1,2,4-triazole coupled with ethyl cyanoacetate to yield a mixture of two geometrical isomers of ethyl 2-(2H-1,2,4-triazol-3-ylhydrazono) cyanoacetate.Recrystallization of the crude coupling mixture from aqueous ethanol gave a single hydra-zone which cyclized predominantly to ethyl 7-amino-1,2,4-triazolo[5,1-c]-1,2,4-triazine-6-carboxylate in acid conditions and 6-cyano-1,2,4-triazolo[ 5,1-c]-1,2,4~triazin-7(4H)-one under basic conditions. The nature of the cyclizing medium also controlled the cyclization of .the (pyrazol-ylhydrazono) cyanoacetate hut the corresponding (tetrazol- ylhydrazono) cyanoacetate gave only ethyl 7-aminotetrazolo[ 5,1-cJ-1,2,4- -triazine-6-carboxylate. 2-( 2H-1,2,4-Triazol-3-:ylhydrazono) malonitrile cyclized unambiguously to 7-amino-6-cyano-1,2,4-triazolo-[ 5,1-c]-1,2,4- triazine. Drastic hydrolysis of ethyl 2-(2H-1,2,4-triazol-3-yllhydrazono)-cyanoacetate, ethyl 7-amino-1, 2,4-triazolo[ 5,1-c]-1,2,4-triazine-6-carboxylate, 6-cyano-1,2,4-triazolo[ 5,1-c]-1,2,4-triazin-7{ 4H)-one and 7-amino-6- cyano-1,2,4-triazolo[5,1-c]-1,2,4-triazine gave a hydrate of 1,2,4-triazo1o[5,1-c ]-1,2,4-triazin-7(4H)-one. Mass spectral fragmentations of 7-aminoazolo-[5,1-c]-1,2,4-triazinesconfirm that the azole ring is more stable than the 1,2,4-triazine ring on electron impact.

Preparation, optimisation and characterisation of lyophilised rapid disintegrating tablets based on gelatin and saccharide

Despite recent success, rapidly disintegrating lyophilized tablets still face problems of low mechanical strength and higher disintegration times resulting in poor patient compliance. The aim of the current work was to carry out a systematic study to understand the factors controlling mechanical properties of these formulations. The work investigated the influence of two bloom strengths of gelatin: low (60 bloom) and high (225 bloom) at different stock solution concentrations (2, 5, 7.5, and 10 %w/w). This was followed by investigation of addition of five saccharides (xylitol, glucose, trehalose, maltotriose and mannitol) at varied concentration range (10-80 %w/w) to decipher their influence on disintegration time, mechanical and thermal properties of the formulation. The results indicated that the disintegration time of the tablets dramatically decreased by decreasing the concentration and bloom strength of gelatin in the stock solution. However the mechanical properties of the tablets were mainly influenced by the concentration of gelatin rather than the bloom strength. The addition of saccharides resulted in enhancement of tablet properties and was concentration dependent. All the saccharides improved the fractubility of the tablets significantly at high concentration (equal or higher than 40% w/w). However, only high concentration (equal or higher than 40% w/w) of trehalose, maltotriose and mannitol significantly enhanced the hardness. Additionally, mannitol crytallised during freeze drying and consequently produced elegant tablets, whilst the other saccarides exhibited lyoprotectant activity as they were able to retain amorphous status. Based on the above findings, an optimized formulation was also successfully developed and characterized to deliver 100 microg dose of Clonidine HCl.

The synthesis and application of room temperature ionic liquids

This research project is concerned with the development and use of eco-friendly reaction media for a variety of organic transformations in the preparation of organic chemicals with potential pharmaceutical applications. These chemicals will then be investigated for their anti-cancer, anti-bacterial and anti-inflammation properties. In this project, different methods were used to synthesize various kinds of ionic liquids. Some new ionic liquids were prepared. In addition, Knoevenagel condensation reactions were investigated in RTILs. For the first time, some neutral ionic liquids such as [BMIM]+[BF4]-, [MeOEtMIM]+[CF3COO]- acted as both catalysts and solvents to promote Knoevenagel reactions. All these experiments indicated that RTILs have a great potential as alternative solvents in synthetic chemistry. Furthermore, nucleoside chemistry is an important research area in drug discovery. Various chemical modified nucleosides have therapeutic activities. However, these compounds usually have poor solubility in common organic solvents. RTILs such as [MeOEtMIM]+[CH3SO3]- have good dissolving capability for these chemicals. A range of thio-substituted nucleobases and nucleosides with potential pharmaceutical applications have been synthesized in several RTILs. These chemicals will then be investigated for their anti-cancer properties.

Computer-aided design, synthesis and screening of potential anti-microbial compounds

The work presented in this thesis falls into three main categories: The design and synthesis of potential anti-tuberculosis drugs targeting a mycobacterial esterase and the enzyme dUTPase; synthesis and anti-microbial SAR studies on a set of carboxamidrazones; synthesis and anti-microbial SAR studies on a set of thiosem icarbazones.

Design, synthesis and development of PDE5 inhibitors

This research project is concerned with the design, synthesis and development of new phosphodiesterase 5 (PDE5) inhibitors with improved selectivities and lower toxicities. Two series of a 5 member and a 6 member ring fused heterocyclic compounds were designed, and synthesized. By alteration of starting materials and fragments, two virtual libraries, each is consisted of close to hundred compounds, were obtained successfully. The screening of sexual stimulation activity with rabbits demonstrated both groups of compounds were able to stimulate rabbit penile erection significantly. The following toxicity studies revealed 2-(substituted-sulfonylphenyl)-imidazo [1,5-a]-1,3,5-triazine-4-(3H)-one group possessed an unacceptable toxicity with oral LD50 about 200mg/kg; while 2-(substituted-sulfonylphenyl)-pyrrolo[2,3-d]pyrimidin-4-one group showed an acceptable toxicity with oral LD50 over 2000mg/kg. The continued bioactivity studies showed yonkenafil, the representative of 2-(substituted-sulfonylphenyl)-pyrrolo[2,3-d]pyrimidin-4-one group, has a better selectivity towards PDE5 and PDE6 than sildenafil and a better overall profile of sexual stimulation on animals than sildenafil. Chronic toxicity studies of yonkenafil further confirmed yonkenafil did not cause any serious side effect and damage on animal models and most actions were explainable. Based on evidences of the above studies, yonkenafil were recommended to enter clinical trials by the regulation authority of China, SFDA. Currently yonkenafil has been through the Phase I clinical trials and ready to progress into Phase II. Hopefully, yonkenafil will provide an alternative to the ED patients in the future.