This project investigated the development of novel anticancer agents with good efficacy and selectivity. Cryptolepine is an alkaloid found in the roots of West African climbing shrub species including Cryptolepis triangu- laris and Cryptolepis sanguinolenta. Cryptolepine is 5-methyl-1oH-indolo [3, 2-b]quinolone, and was first identified as an antimalarial agent, but also acts as an anti-cancer agent by intercalating into DNA and also inhibiting topoi- somerase ll and other key enzymes. Studies elsewhere have shown the mode of action of cryptolepine in vitro appears to be unaffected by drug re- sistance mechanisms identified. In this project a number Cryptolepine ana- logues have been made, modifying key positions in order to enhance DNA binding. The aim of this study was to attach halogens (F, Cl, Br and I) and alkyl amino or amido side chains at the 11-position and then test these mole- cules for anticancer activity. It was anticipated that these nitrogen containing side chains might interact with the sugar-phosphate backbone of DNA to give improved binding and hence interfering with topoisomerase II and related enzymes such as helicase and hence enhancing cytotoxicity. Fluorescence microscopy was used to investigate whether the derivatives reach the cell nucleus. In conclusion, these studies have shown that novel amino- and halogenated cryptolepine analogues have greater in vitro cytotoxicity than the parent compound and are important lead compounds in the development of novel potent and selective indoloquinone anti-neoplastic agents.

The Tyrrell group has, in recent years, developed a strong interest in the chemical synthesis and biological activity of the hop bitter acids lupulone 1, humulone 2 and their naturally occurring congeners, 1 a-d and 2a-d. Previous work focused on investigating the chemistry of the a-acid humulone 2, demonstrating the propensity of this compound to have a cytotoxic effect against the SK-MES lung cancer cell line and the MCF-7 breast cancer cell line. This thesis documents the recent research concerning the synthesis, the anticancer and antibacterial activity of the ß-acid lupulone 1, its naturally occurring congeners 1 a-d and further, novel, non-naturally occurring compounds. We resumed the group's previous collaboration with the Golston and Pirianov research group at St Georges, where the anticancer studies were performed. The synthesis centred on a Friedel-Crafts acylation of phloroglucinol 32, followed by a C-trialkenylation reaction between the acylphloroglucinol and an allyl bromide. We conducted experiments that focused upon the choice of base and solvent for the trialkenylation reaction, concluding that liquid ammonia as both the base and solvent offered the most efficient route to the ß-acids. It was shown that aliphatic allylic bromides lend themselves to the' reaction, . although some derivatives require purification by column chromatography in addition to recrystallisation. In contrast, aromatic allyl bromides did not participate in the alkenylation reaction. We further investigated an alternate G-alkenylation reaction involving the di¬lithiation of 1,3,5-trimethoxybenzene. We discovered that by including copper (I) iodide in the reaction, prenyl bromide 23 and allyl bromide 41 could be successfully coupled. VVe also discovered that our 2nd generation ß-acids 42a-g could participate in a ring-closing metathesis reaction, forming novel spirocyclic compounds 50a-b. Our antibacterial studies showed that ß-acids are effective against Gram-positive bacteria, but not Gram-negative bacteria in accordance with published observations. We took our investigations further and found that ß-acids are effective against mutlidrug-resistant 'Staphylococcus aureus', even where the commercially available ciprofloxacin 67 was not.

Resveratrol is a natural polyphenol present in many plant species and derived foods including grapes and red wine. It has been shown to possess chemotherapeutic properties in animal cancer models as well as many biological effects in vitro. In this study, the effects of three resveratrol metabolites including resveratrol-3-0-D-glucuronide, resveratrol-4'-O-D-glucuronide and resveratrol-3-0-D-sulphate on the growth of colon cancer cell lines have been investigated. The growth inhibitory effects of resveratrol, piceatannol, pterostilbene and the glucuronide and sulphate metabolites on Caco-2, CCL-228 and HCT-116 cells were assessed using the neutral red and MTT assays. Resveratrol and its metabolites inhibited the growth of cells with ICso values in the range of 9.8-31 f.lM whilst piceatannol and pterostilbene in the range of 21.7-29.6f.lM and 5-34.5f.lM respectively. Apoptotic assessment by DAPI staining, Z-VAD-FMK pre-treatment and percentage of cells in sub-Gl by FACS all revealed the absence of apoptosis. Treatment of differentiated Caco-2 mono layers showed that resveratrol was capable of inhibiting the growth of cells following treatment on the apical but not basolateral membrane and the effects were less profound with the metabolites. Only high concentrations (500f.lM) of metabolites (not used in any ofthe growth studies) appeared to be toxic to normal cells as measured by a haemolysis assay. Resveratrol was capable of causing S phase arrest in all 3 cell lines at 30f.lM whilst the two glucuronides caused GO/GI arrest in Caco-2 and CCL-228 cells only. Resveratrol-3-0-D-sulphate had no effect on the cell cycle. Growth inhibition caused by resveratrol and its two glucuronides was reversed by the addition of an AMP kinase inhibitor (compound C) or an adenosine A3 receptor antagonist (MRS-119l). Treatment with the highly selective A3 receptor agonist, 2CI-m-MECA caused growth inhibition and the A3 receptor was 'detected in all 3 cell lines. Levels of eyelin D 1 as measured by western blot were significantly reduced at higher concentrations (lOOf.lM) but p-AMPK was not reliably increased in all cases. Resveratrol glucuronides were shown to inhibit the growth of three colon cancer cell lines by GO/G 1 arrest and depletion of eyelin D 1. These findings strongly suggest a role of the adenosine A3 receptor in the observed inhibition therefore, providing a novel target for resveratrol and its metabolites.

Enzymes such as aromatase, 17[beta]-hydroxysteroid dehydrogenase [types 1 (17[beta]-HSD1) and 3 (17[beta]-HSD3)] and estrone sulfatase (ES) are all involved in the biosynthesis of steroids via the steroidal cascade. The inhibition of these enzymes may lead to a reduction in the levels of steroids present, thereby leading to a decrease in the stimulation of hormone-dependent tissues, in particular, hormone-dependent breast and prostate cancers. This approach has proved to be successful in postmenopausal women where the use of aromatase inhibitors has led to the decrease in tumour yield and has thus led to the treatment of the disease. Within the current study, the synthesis and biochemical evaluation of a number of compounds of varying structural features has been undertaken, in particular, the synthesis of sulfonate derivatives of 4-hydroxyphenyl ketone - the parent compound having already been shown to be a potent inhibitor of 17[beta]-HSD3 (with good specificity towards 17[beta]-HSD3) and the synthesis of a range of alkyl and cycloalkyl esters of steroids [in particular, testosterone (T) dehydroepiandrosterone (DHEA) and estrone (E10] as probes of the active sites of the HSD family of enzymes. The results show that the sulfonate (methanesulfonate and trifluromethanesulfonate) derivatives of 4-hydroxyphenyl ketone-based compounds were found to possess weak inhibitory activity against all three HSD enzymes considered (namely, 17[beta]-HSD1, 17[beta]-HSD3 and 3[beta]-HSD) in comparison to the parent 4-hydroxyphenyl ketone-based compounds. For example, within the methanesulfonate derivatives, methane sulfonic acid (4-cyclohexane carbonyl)-phenyl ester (164) was found to be the most potent inhibitor against 17[beta]-HSD3, however, it possessed ~30% inhibitory against this enzyme at an inhibitor concentration of 100[mu]M. Against 17[beta]-HSD1, the most potent compound within the same range was also compound 164 which pssessed ~45% inhibitory activity under similar conditions. Within the trifluromethane sulfonate derivatives, the most potent compounds proved to be extremely weak inhibitors of 17[beta]-HSD3, however, against 17[beta]-HSD1, the most potent compound was trifluromethane sulfonic acid 4-benzoyl-phenyl ester (180) which possess 43% inhibitory activity. The molecular modeling of these compounds within representations of the active sites of 17[beta]-HSD1 and 17[beta]-HSD3 shows that the lack of inhibitory activity is due to steric hindrance, in particular, the sulfonate moeity undergoes steric hindrance with groups at the active site which is close to the C(17) area of the natural substrate. The synthesis of the esters of T, DHEA and E1 and the subsequent biochemical evaluation of these compounds resulted in an interesting structure-activity relationship. In general, the compounds based on DHEA were found to be potent inhibitors of 17[beta]-HSD3 with weak inhibitory activity against 17[beta]-HSD1 and 3[beta]-HSD. For example, DHEA acetate (196) was found to possess an IC[sub]50 value of 0.74[mu]M in comparison to the most potent standard, namely 1-(4hydroxy-phenyl)-nonan-1-one (139) which was found to possess an IC[sub]50 value of 12.32[mu]M - this compound was found to possess good selectivity as it possessed ~40% and ~25% inhibitory activity against 17[beta]-HSD1 and 3[beta]-HSD respectively at an inhibitor concentration of 100[mu]M. The esters of E1 and T proved to be weaker inhibitors in comparison to the esters based on DHEA, however, the E1-based esters also showed some selectivity towards 17[beta]-HSD3. For example, E1 hexanoate (216) possessed an IC[sub]50 value of 37.28[mu]M and possessed 45% and 35% inhibitory activity against 17[beta]-HSD1 and 3[beta]-HSD respectively at an inhibitor concentration of 100[mu]M. The modelling of these compounds (using representations of the active sites of 17[beta]-HSD1 and 17[beta]-HSD3) showed that the lack of inhibitory activity was due to steric interactions between the inhibitors and groups within the active site. As such, these compounds proved to be extremely useful probes of the active sites of 17[beta]-HSD1 and 17[beta]-HSD3 and have further enhanced the models used in the design of these compounds.

It has been shown that the majority of benign prostatic hyperplasia (BPH) and prostate cancers are dependent on androgen production within the body. The biosynthesis of androgens is catalysed by different enzymes however one of the enzymes, 17 beta-hydroxysteroid dehydrogenase type 3 (17 beta-HSD3), converts the C(17)=O carbonyl moiety of androstenedione (1l4-dione) to the corresponding C(17)-OH hydroxyl group of testosterone (T). It has been hypothesised that inhibition of 17 beta-HSD3 may cause a decrease in the level of androgens which in turn leads to a reduction in the genesis of androgen-dependent prostatic diseases. The utilisation of enzyme inhibition as a therapeutic agent, in the treatment of breast cancer, has been tested on postmenopausal women by using aromatase inhibitors (e. g; exemestane, anastrazole and latrazole). This approach has proved to be successful and the impact of enzyme inhibition was led to a reduction in cancer growth. This process has now found a clinical application. From molecular modelling studies it was postulated that any potential inhibitor of 17 beta-HSD3 should contain a carbonyl moiety, mimicking the C(17)=O of the natural substrate, as well as an aromatic ring adjacent to the carbonyl group. With these criteria in mind results from our laboratories showed that from a library of candidates those based upon 4-hydroxyphenyl ketones showed some potential. The main focus of this prestn study was to fine tube the enzyme inhibitor analogues and hence optimise inhibitory activity of 4-hydroxyphenyl ketones. We have successfully synthesised a range of novel derivatives of 4-hydroxyphenyl ketones such as the 4-methanesulfonate and 4-acetate ester derivatives. In general, the reactions have proceeded very well with the yields ranging from 65% to 88% and 91% to 97% respectively. The results of biochemical evaluation studies suggested that the acetate ester derivatives, in particular compounds (149) and (150) exhibited good inhibitory activity against 17 beta-HSD type 3 of about 40% compared to standard inhibitors such as 7-hydroxyflavone and baicalein which resulted in about 13% and 14% inhibitory activity respectively. In addition a range of non-steroidal B, C, D ring mimics of the natural substrate of 17 beta-HSD type 3 were synthesised in good yields (65% to 85%). The biochemical evaluation of these compounds also showed good inhibitory activity; in fact compound (107) exhibited about 43% inhibition in comparison to the above standards which had inhibition of about 25% and 31 % respectively. In conclusion we have successfully synthesised and biochemically evaluated a number of enzyme inhibitors for the enzyme 17 beta-HSD type 3. The two types of active inhibitors were structurally dissimilar suggesting that they may have different modes of binding. This outcome requires further investigation in order to establish and identify how this inhibition is taking place.

Androgens play an important part in the initiation and progression of hormone-dependent prostate and breast cancer. These types of cancers can be treated by androgen ablation therapy. However, androgen ablation is associated with short (2-3 years) remission of the disease. Therefore therapies that inhibit the systemic biosynthesis of androgens, by targeting the P450 enzymes (CYP17 and 17-[beta]HSD type 3) which catalyse androgen biosynthesis, may represent a rational approach in the treatment of androgen-dependent cancer. Inhibitors of the enzyme CYP17: ketoconazole and liarozole, have been shown to decrease tumour cell adhesion to the endothelium and expression of adhesion molecules. The adhesion of cancer cells to the endothelium is an important preliminary event that underlies cancer matastasis. Within the this study, the development of assays for the enzymes; CYP17 and 17[beta]-HSD3 and the evaluation of a series of compounds which were designed to inhibit these enzymes have been considered. The preliminary screening of the compounds showed good inhibition of 17[alpha]=OHase and 17, 20 lyase components of the CYP17 enzyme in comparison to the reference drug, ketoconazole (KTZ). The IC[sub]50 of compounds 31, 34, 38, 41, 48 and 51 and KTZ was calculated as 14.40 [Mu]M, 5.82 [Mu]M, 0.18 [Mu]M, 1.35[Mu]M, 1.21[Mu]M, 0.50[Mu]M and 5.65[Mu]M respectively. However, only a few of the compounds designed to inhibit 17[beta]-HSD3 showed ap potent inhibitory activity. Compound 132 showed the highest percentage inhibition (40.51 [plus or minus] 0.14%) of 17[beta]- HSD3 activity when compared to the reference drugs, 7-hydroxy flavone (12.90 [plus or minus] 0.31%) and biacalein (13.66 [plus or minus] 0.31%). CYP17 inhibitors did not have any cytoxic effect on human cancerous and non-cancerous cell lines. The adhesion of DU145, PC3 and MCF7 to a non-stimulated HUVEC monolayers was decreased from 100 [plus or minus] 0.01% cell adhesion to 60.93 [plus or minus] 3.95%, 65.79 [plus or minus] 9.39% and 65.12 [plus or minus] 4.04% by compounds 38. 48 and 51 respectively in the absence of tumour necrosis factor alpha (THF-[alpha]). Similarly, compounds 38, 48 and 51 showed the highest anti-adhesion effect of DU145 on stimulated HUVEC monolayers (69.85 [plus or minus] 2.51%) cells respectively. Flow cytometry and immunostaining of intracellular adhesion molecules showed that CYP17 inhibitors did not have any effect on the expression of ICAM-1. In conclusion, the synthesised compounds were found to be good indicators of the CYP17 enzyme with no cytoxic and better anti-adhesion effects when compared to KTZ. Thus, these compounds can be further investigated as a therapeutic strategy against hormone-dependent prostate and breast cancer.

The research work presented in this thesis deals with the anticancer activity of four medicinal plants: 'Caralluma tuberculata' (Asclepiadaceae), 'Fagonia indica' (Zygophyllaceae), 'Solanum surattense' (Solanaceae) and 'Arisaema utile' (Araceae) that originate from the North West and Himalayan regions of Pakistan. Through a bioactivity-guided fractionation approach, the crude and resultant organic fractions were tested on cultured breast cancer cells (MCF-7 and MDA MB-468) and colorectal carcinoma cells (Caco-2) in vitro. Five new compounds out of seven in total were isolated from potent fractions of the new medicinal plants using repeated flash column chromatography. Structural elucidation was carried out through a series of spectroscopic experiments (1-D and 2-D NMR, GC-MS, LC-MS). SIngle crystal X-ray structure was determined using X-ray crystallography for the crystalline compounds, which showed a defraction pattern. The apprent IC[sub]50 for compounds (1-6) were estimated from serial dilutions of eight concentrations (0.78-100 [mu]M) of each compound, tested against breast and colon cancer cell lines, using two cell viability assays (MTT and neutral red uptake assays) for 24 h and 48 h treatments. Two new steroidal glycosides, acylated pregnane (1) and acylated androstane (2) glycosides, isolated from the ethyl acetate fraction of 'Caralluma tuberculata' showed highly significant (P<0.001) percentage growth inhibition in Caco-2 cells (IC[sub]50) 1.56-6.25 [mu]M) and MCF-7 cells (IC[sub]50 6.25 - 25 [mu]M), however, oestrogen independent cancer cells (MDA MB-468) were less responsive with IC[sub]50 25 - 50 [mu]M. These steroidal glycosides induced apoptosis in cancer cells as measures of cytoxic activity (NRU, PARP clevage, DNA ladder) on MCF-7, MDA MB-468 and Caco-2 cells were inhibited by pre-treatment with the pan-caspase inhibitor (Z-VAD-FMK). Another pregnane glycoside (3), isolated for the first time from 'Fagonia indica', was found to be more potent in suppressing cell growth (IC[50] 6.25-25 [mu]M), in oestrogen negative breast cancer cells (MDA MB-468,) as compared to oestrogen positive cancer cells (MCF-7). Although a cleaved PARP (89kDa) was detected by Western blotting, cytomorphological alterations and in cells pre-treated with a pan-caspase inhibitor (NRU assay), indicated that the necrosis mode of cell death is more likely. Moreover, three esters: hexadecanoic acid ethyl ester (4), phtalic acid 1-(1, 1-dimethyl-pentyl) ester 2-(2-ethyl-dec-5-enyl) ester (5) from chloroform fraction of 'Solanum surattense', and 5-Oxo-19-propyl-docosanoic acd methyl ester (6) from 'Arisaema utile', showed a highly significant )p<0.001) decrease in cell numbers for MDA MB-468 and Caco-2 cells with apparent IC[50] 6.25-12.5 [mu]M in cell viability assays (MTT and NRU) after 48 h treatment, while MCF-7 cells were less responsive (IC[sub]50 25 [mu]M). Compunds 5 and 6 (first report from a natural source) did not restrict the growth inhibition in MCF-7 and Caco-2 cells, pre-treated with Z-VAD-FMK, which indicated less involvement of Capase-dependent apoptosis, while DAPI staining and Western blots (cleaved-PARP) showed characteristics of apoptosis that suggested the possibility of aponecrosis phenomenon of cell death. In preliminary screening (Western blot and DNA ladder assays), compounds (1-6) were not toxic to normal human cells (HUVEC and U937) and indicated some selctivly between malignant and normal cells.

Despite the significant improvement in CML treatment since the introduction of Glivec, resistance to synthetic Tyrosine Kinase Inhibitors is emerging as a major limitation. 50%-90% of patients acquire resistance through point mutations that might span the ABL1 kinase domain, while 10%-50% may resist treatment through other mechanisms. Qatar with its limited 1.6 million inhabitants and 15 CML patients diagnosed yearly has the highest rate of disease resistance to Glivec treatment. Through a prospective study, this project examined the rate of resistance to Glivec and the different mechanisms that could be responsible for this problem. Thus over a period of 5 years, 26 patients were treated with Glivec as a front line therapy and their response to treatment was monitored objectively according to the ELN 2006 response criteria. 12 of the 26 patients responded optimally to treatment, while 14 patients did not (9 failed the treatment and 5 responded sub-optimally), setting the resistance rate to about 54% which is the highest reported worldwide to date. None of our patients showed any of the reported mutations that are known to confer resistance to Glivec. However in one patient, direct sequencing showed a Single Nucleotide Polymorphism (SNP) that might function as a resistance inflecting mutation. In another patient who resisted treatment a transient insertion of three nucleotides (AAG) at position 1432 which adds an amino acid Lysine to position 356 of the catalytic domain of ABL1 was revealed. To our knowledge this transit insertion of nucleotides and amino acid addition was not reported before. 7 patients showed Additional Chromosomal Abnormalities (ACA) at time of resistance, while 2 patients were intolerant to treatment and 3 had no identifiable cause for their resistance. Patient compliance was ruled out as a cause of resistance in our patients, however the quality of service providing was audited and certain financial and clinical management issues were addressed during the course of this study.