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.

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.

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.