Flavones and related compounds as inhibitors of protein tyrosine kinases

Aberrant tyrosine protein kinase activity has been implicated in the formation and maintenance of malignancy and so presents a potential target for cancer chemotherapy. Quercetin, a naturally occuring flavonoid, inhibits the tyrosine protein kinase encoded by the Rous sarcoma virus but also exhibits many other effects. Analogues of this compound were synthesised by the acylation of suitable 2-hydroxyacetophenones with appropriately substituted aromatic (or alicyclic) acid chlorides, followed by base catalysed rearrangement to the 1-(2-hydroxyphenyl)-3-phenylpropan-1,3-diones. Acid catalysed ring closure furnished flavones. The majority of the 1-(2-hydroxyphenyl)-3-phenylpropan-1,3-diones were shown by NMR to exist in the enol form. This was supported by the crystal structure of 1-(2-hydroxy-4-methoxyphenyl)-3-phenylpropan-1,3-dione. In contrast, 1.(4,6-dimethoxy-2-hydroxyphenyl)-3-phenylpropan-1,3-dione did not exhibit keto-enol tautomerism in the NMR spectrum and was shown in its crystal structure to assume a twisted conformation. Assessment of the biological activity of the analogues of quercetin was carried out using whole cells and the kinase domain of the tyrosine protein kinase encoded by the Abelson murine leukaemia virus, ptab150 kinase. Single cell suspension cultures and clonogenic potential of murine fibroblasts transformed by the Abelson Murine leukaemia virus (ANN-1 cells) did not indicate the existence of any structure activity relationship required for cytotoxicity or cytostasis. No selective toxicity was apparent when the `normal' parent cell line, (3T3), was used to assess the cytotoxic potential of quercetin. The ICS50 for these compounds were generally in the region of 1-100M. The potential for these compounds to inhibit ptab150 kinase was determined. A definite substitution requirement emerged from these experiments indicating a necessity for substituents in the A ring or in the 3-position of the flavone nucleus. Kinetic data showed these inhibitors to be competitive for ATP.

Inhibitors of lipolysis in tumor-induced cachexia

The MAC16 tumour produces a factor which exhibits lipid-mobilizing activity in vitro in addition to causing extensive depletion of host lipid stores. The mechanism of the anti-lipolytic effect of two anti-cachectic agents, eicosapentaenoic acid, an ω-3 polyunsaturated fatty acid (PUFA), and N-(3-phenoxycinnamyl)acetohydroxamic acid (BW A4C), a 5-lipoxygenase inhibitor, has been investigated. These two agents reduce tumour growth and reverse the weight loss which accompanies transplantation of the MAC16 murine colon adenocarcinoma into NMRI mice. Mice transplanted with the MAC16 tumour exhibited weight loss which was directly proportional to the serum lipolytic activity measured in vitro up to a weight loss corresponding to 16% of the original body weight. After this time, an inverse relationship between weight loss and lipolytic activity was observed. Body composition analysis revealed a large decrease in body fat relative to other body compartments. The anti-tumour/anti-cachectic effect of EPA did not appear to be due to its ability to inhibit the production of prostaglandin E2. The MAC16 lipolytic factor increased adenylate cyclase activity in adipocyte plasma membranes in a concentration-dependent manner. EPA inhibited the production of cAMP attributed to this lipid-mobilizing factor. EPA produced alterations in Gi , the guanine nucleotide binding protein which mediates hormonal inhibition of adenylate cyclase, in addition to altering cAMP production in adipocyte plasma membranes in response to hormonal stimulation. The alterations in adenylate cyclase activity were complex and not specific to EPA. EPA stimulated adenylate cyclase activity when in a relatively high fatty acid : membrane ratio and inhibited activity when this ratio was lowered. The inhibitory effect of EPA on adenylate cyclase activity may be the underlying mechanism which explains its anti-lipolytic and anti-cachectic effect. The inability of the related ω-3 PUFA, docosahexaenoic acid (DHA), to inhibit cachexia may be due to a difference in the metabolic fates of these two fatty acids. BW A4C inhibited lipolysis in isolated adipocytes which suggests that this compound may possess the potential for an anti-cachectic effect which is independent of its inhibitory effect on tumour growth.

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.

Local and systemic delivery of a novel group of inhibitors of transglutaminase enzyme: A potential approach for treating of catheter-related complications and liver fibrosis

The present thesis investigates targeted (locally and systemically) delivery of a novel group of inhibitors of enzyme transglutaminases (TGs). TGs are a widely distributed group of enzymes that catalyse the formation of isopeptide bonds between the y-carboxamide group of protein-bound glutamines and the a-amino group of protein-bound lysines or polyamines. The first group of the novel inhibitors tested were the tluorescently labelled inhibitors of Factor XIIIa (FXIIIa). These small, non-toxic inhibitors have the potential to prevent stabilisation of thrombi by FXIIIa and consequently increase the natural rate of thrombolysis, in addition it reduces staphylococcal colonisation of catheters by inhibiting their FXIIIa¬mediated cross-linking to blood clot proteins on the central venous catheter (CVCs) surface. The aim of this work was to incorporate the FXIIIa inhibitor either within coating of polyurethane (PU) catheters or to integrate it into silicone catheters, so as to reduce the incidence of thrombotic occlusion and associated bacterial infection in CVCs. The initial work focused on the incorporation of FXIIIa inhibitors within polymeric coatings of PU catheters. After defining the key characteristics desired for an effective polymeric-coating, polyvinylpyrrolidone (PVP), poly(lactic-co-glycolic acid) (PLGA) or their combination were studies as polymers of choice for coating of the catheters_ The coating was conducted by dip-coating method in a polymer solution containing the inhibitor. Upon incubation of the inhibitor-and polymer-coated strips in buffer, PVP was dissolved instantly, generating fast and significant drug release, whilst PLGA did not dissolve, yielding a slow and an insufficient amount of drug release. Nevertheless, the drug release profile was enhanced upon employing a blend solution of PVP and PLGA. The second part of the study was to incorporate the FXIIIa inhibitor into a silicone elastomer; results demonstrated that FXIIIa inhibitor can be incorporated and released from silicone by using citric acid (CA) and sodium bicarbonate (SB) as additives and the drug release rate can be controlled by the amount of incorporated additives in the silicone matrix. Furthermore, it was deemed that the inhibitor was still biologically active subsequent to being released from the silicone elastomer strips. Morphological analysis confirmed the formation of channels and cracks inside the specimens upon the addition of CA and SB. Nevertheless, the tensile strength, in addition to Young's modulus of silicone elastomer strips, decreased constantly with an increasing amount of amalgamated CA/ SB in the formulations. According to our results, incorporation of FXIIIa inhibitor into catheters and other medical implant devices could offer new perspectives in preventing bio-material associated infections and thrombosis. The use of tissue transglutaminase (T02) inhibitor for treating of liver fibrosis was also investigated. Liver fibrosis is characterized by increased synthesis and decreased degradation of the extracellular matrix (ECM). Transglutaminase-mediated covalent cross-linking is involved in the stabilization of ECM in human liver fibrosis. Thus, TG2 inhibitors may be used to counteract the decreased degradation of the ECM. The potential of a liposome based drug delivery system for site specific delivery of the fluorescent TG2 inhibitor into the liver was investigated; results indicated that the TG2 inhibitor can be successfully integrated into liposomes and delivered to the liver, therefore demonstrating that liposomes can be employed for site-specific delivery of TG2 inhibitors into the liver and TG2 inhibitor incorporating liposomes could offer a new approach in treating liver fibrosis and its end stage disease cirrhosis.

Corrosion inhibitors for the rehabilitation of reinforced concrete

Four corrosion inhibitors namely sodium nitrite, sodium monofluorophosphate, ethanolamine and an alkanolamine-based mixture were studied by immersing mild steel bars for 42 days in model electrolytes of varied pH and chloride concentration which were intended to simulate the pore solution phase present within carbonated and/or chloride-contaminated concrete. Site trials were carried out on sodium monofluorophosphate and the alkanolamine-based inhibitor to study their depth of penetration into concrete. The influence of various carbonating atmospheres on the pore solution chemistry and microstructure of hydrated cement paste was investigated. Physical realkalisation of carbonated cement paste and a calcium nitrite-based corrosion rehabilitation system for chloride-contaminated cement paste were investigated by monitoring ionic transport within the pore solution phase of laboratory specimens. The main findings were as follows: 1,Sodium nitrite, sodium monofluorophosphate, ethanolamine and the alkanolamine-based mixture all behaved as passivating anodic inhibitors of steel corrosion in air-saturated aqueous solutions of varied pH and chloride concentration. 2,Sodium monofluorophosphate failed to penetrate significantly into partially carbonated site concrete when applied as recommended by the supplier. Phosphate and fluoride penetrated 5mm into partially carbonated site concrete treated with sodium monofluorophosphate. 3,The ethanolamine component of the alkanolamine-based inhibitor was found to have penetrated significant depths into partially carbonated site concrete. 4,Carbonating hydrated cement paste over saturated solutions of sodium nitrite resulted in significant concentrations of nitrite in the pore solution of the carbonated paste. Saturated solutions of sodium chloride, ammonium nitrate, magnesium nitrate and sodium dichromate were investigated and identified as alternatives for controlling the relative humidity of the carbonating environment. 5,Hardened carbonated cement paste can by physically realkalised to a limited extent due to the diffusion of hydroxyl ions under saturated conditions. A substantial proportion of the hydroxyl ions that diffused into the carbonated cement paste however, became bound into the cement matrix. Hydroxyl ion concentrations remained below 5mmol/l within the pore solution of the realkalised cement paste. 6, Nitrite ions penetrated significant distances by diffusion within the pore solution of saturated uncarbonated hydrated cement paste.

The neurotoxic effects of enzyme inhibitors at the neuromuscular junction

Current knowledge of the long-term, low dose effects of carbamate (CB) anti-cholinesterases on skeletal muscle or on the metabolism and regulation of the molecular forms of acetylcholinesterase (AChE) is limited. This is largely due to the reversible nature of these inhibitors and the subtle effects they induce which has generally made their study difficult and preliminary investigations were conducted to determine suitable study methods. A sequential extraction technique was used to rapidly analyse AChE molecular form activity at the mouse neuromuscular junction and also in peripheral parts of muscle fibres. AChE in the synaptic cleft involved in the termination of cholinergic transmission was successfully assessed by the assay method and by an alternative method using a correlation equation which represented the relationship between synaptic AChE and the prolongation of extra-cellular miniature endplate potentials. It was found that inhibition after in vivo Carbamate (CB) dosing could not be maintained during tissue analysis because CB-inhibited enzyme complexes decarbamoylated vary rapidly and could not be prevented even when maintained on ice. The methods employed did not therefore give a measure of inhibition but presented a profile of metabolic responses to continual, low dose CB treatment. Repetitive and continual infusion with low doses of the CBs: pyridostigmine and physostigmine induced a variety of effects on mouse skeletal muscle. Both compounds induced a mild myopathy in the mouse diaphragm during continual infusion which was characterised by endplate deformation without necrosis; such deformation persisted on termination of treatment but had recovered slightly 14 days later. Endplate and non-endplate AChE molecular forms displayed selective responses to CB treatment. During treatment endplate AChE was reduced whereas non-endplate AChE was largely unaffected, and after treatment, endplate AChE recovered, whereas non-endplate AChE was up-regulated. The mechanisms by which these responses become manifest are unclear but may be due to CB-induced effects on nerve-mediated muscle activity, neurotrophic factors or morphological and physiological changes which arise at the neuromuscular junction. It was concluded that, as well as inhibiting AChE, CBs also influence the metabolism and regulation of the enzyme and induce persistent endplate deformation; possible detrimental effects of long-term, low-dose determination requires further investigation.

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.

Indwelling catheters and medical implants with FXIIIa inhibitors:a novel approach to the treatment of catheter and medical device-related infections

Central venous catheters (CVCs) are being utilized with increasing frequency in intensive care and general medical wards. In spite of the extensive experience gained in their application, CVCs are related to the long-term risks of catheter sheath formation, infection, and thrombosis (of the catheter or vessel itself) during catheterization. Such CVC-related-complications are associated with increased morbidity, mortality, duration of hospitalization, and medical care cost [1]. The present study incorporates a novel group of Factor XIIIa (FXIIIa, plasma transglutaminase) inhibitors into a lubricious silicone elastomer in order to generate an optimized drug delivery system whereby a secondary sustained drug release profile occurs following an initial burst release for catheters and other medical devices. We propose that the incorporation of FXIIIa inhibitors into catheters, stents, and other medical implant devices would reduce the incidence of catheter sheath formation, thrombotic occlusion, and associated staphylococcal infection. This technique could be used as a local delivery system for extended release with an immediate onset of action for other poorly aqueous soluble compounds. © 2012 Elsevier B.V. All rights reserved.

Imidazolium-based warheads strongly influence activity of water-soluble peptidic transglutaminase inhibitors

New peptidic water-soluble inhibitors are reported. In addition to the carboxylate moiety, a new polar warhead was explored. Depending on the size of its substituents, the newly appended imidazolium scaffold designed to enhance the hydrophilic character of the inhibitors could induce a good inhibition for tissue transglutaminase (TG2) and blood coagulation factor XIIIa (FXIIIa). Correlated with the narrow tunnel that hosts the target catalytic cysteine residue, the various modulations suggest a bent conformation of the ligands as the binding pattern mode. Analogues in the dialkylsulfonium series were also tested and showed specificity for TG2 over FXIIIa. © 2013 Elsevier Masson SAS. All rights reserved.

SGLT2 inhibitors:glucuretic treatment for type 2 diabetes

Sodium glucose co-transporter-2 (SGLT2) inhibitors offer a novel approach to treat diabetes by reducing hyperglycaemia via increased glucosuria. This approach reduces renal glucose reabsorption in the proximal renal tubules providing an insulin-independent mechanism to lower blood glucose. The glucuretics are advanced in clinical development and dapagliflozin has received most extensive study. Once daily dapaglifolozin as monotherapy or as add-on to metformin for 12-24 weeks in type 2 diabetic patients (baseline HbA 8-9%) reduced HbA by about 0.5-1%, accompanied by weight loss (2-3 kg) and without significant risk of hypoglycaemia. Dapagliflozin has reduced insulin requirement and improved glycaemic control without weight gain in insulin-treated patients. A mild osmotic diuresis associated with glucuretic therapy may account for a small increase in haematocrit (1-2%) and reduced blood pressure (2-5 mmHg). Dehydration and altered electrolyte balance have not been encountered. Urinary tract and genital infections increased in most studies with dapagliflozin, but were typically mild - resolving with selfmedication or standard intervention. Thus glucuretics provide a novel insulin-independent approach for control of hyperglycaemia which does not incur hypoglycaemia, promotes weight loss, may reduce blood pressure and offers compatibility with other glucose-lowering agents. © 2010 The Author(s).