Theses (MSc. Analytical & Pharmaceutical Chemistry)

Andrographolide (AGP) is the major ent-labdane diterpenoid isolated from Andrographis paniculata (known locally in Malaysia as Hempedu Bumi). AGP was found to exhibit neuroprotective effects by inhibiting inflammation and oxidative stress. As such, AGP could act as a novel alternative for the treatment of neurological disorder. However, its oral bioavailability is reduced due to inactivation by liver glutathione S-transferase (GST). To tackle this shortcoming, a series of 12,13-dihydroandrographolide (HAGP) analogues were semisynthesised in the previous project where the C12-C13 double bond was reduced to avoid glutathione (GSH) conjugation. Besides that, substituted benzylidene moiety was introduced at C-3 and C-19 hydroxyl groups to increase the lipophilicity of the compounds for better blood-brain-barrier penetration. Among the analogues, 3,19-(3-ethoxy,4-hydroxylbenzylidene)-12,13-dihydroandrographolide (HAGP34) and 3,19-(3-flurobenzylidene)-12,13-dihydroandrographolide (HAGP38) showed better activity in the initial bioassay screening against pheochromocytoma (PC12) cells. In view of this, both compounds were selected to evaluate their in vitro pharmacokinetics and neuroprotective properties in this study. HAGP34 and HAGP38 were shown to avoid GSH conjugation (phase II metabolism). However, in the rat microsomes metabolism experiment to mimic Phase I metabolism in the liver, AGP (intrinsic clearance, CLint = 30 μL/min/mg) was found to be more stable than HAGP34 (CLint = 67 μL/min/mg), but less stable than HAGP38 (CLint = 16 μL/min/mg). These two in vitro pharmacokinetics studies evealed that HAGP38 was more stable than AGP in phase I and phase II metabolism. HAGP38 and AGP showed comparable reactive oxygen species (ROS) inhibition in rat pheochromocytoma cells (PC12), where the percentages of inhibition were 18% and 23% respectively at 10 μM. In mouse microglial cells (BV2), AGP exhibited strong nitric oxide (NO) inhibition where its IC50 value was 5 μM. When compared with HAGP38, the NO inhibition was only 17% at 5 μM. The Trans-AM assay revealed that HAGP38 inhibited NO production via disruption of NF-κB signalling pathway, similar to AGP as reported in the literature. On the other hand, HAGP34 showed weak ROS and NO inhibition in both cell lines. These accumulating findings revealed that the C12-C13 double bond or C-3 and C-19 hydroxyl groups of AGP may play strong roles as the pharmacophore of anti-inflammatory and antioxidant activities. Although the biological activities of HAGP38 were not as potent as than AGP, its improved in vitro pharmacokinetics may give better bioavailability compared to AGP. Therefore, HAGP38 is may be a promising molecule that should be further tested in animal models.

The discovery of new therapeutic agents for inflammation has attracted more attention in recent years. Vanilloids are abundantly present in plants belonging to the Zingiberaceae family, contain a vanillyl group (3-methoxy-4-hydroxy-benzyl group), and possess a wide array of biological properties. Vanilloids contain important pharmacophores, vanillyl moiety; α,-unsaturated carbonyl group and alkyl side chain. However, open chain α,-unsaturated carbonyl groups are known as "promiscuous" because these are not selective in binding to receptors that may result in false-positive activities. In addition the presence of free higher alkyl chain make the compounds very flexible, which may result in decreased or/and non-selective activity. Therefore, in this research the new N-vanillylcyclohex-1-ene-1-caboxamide (1), and N-vanillylcyclopent-1-ene-1-carboxamide (2) were synthesised. These compounds retain the essential pharmacophore features of vanilloids. The essential pharmacophore features in 6-Shogaol are vanillyl group, α,-unsaturated carbonyl group and alkyl side chain containing 6 carbons. The novelty in these compounds is that; α,-unsaturated double bond with respect to a keto group was made endocyclic in 5- and 6- membered rings. The compounds were purified by column chromatography and characterised by 1H-NMR, 13C-NMR, FTIR and Mass Spectra. The toxicity and anti-inflammatory activity of compounds were carried out on RAW 264.7 mouse macrophages and BV2 mouse microgilial cell lines. These two cell lines are most widely studied cell lines to test the anti-inflammatory activity of vanilloids. The concentration range of compounds used iv in this study was 5 - 250 μM. The compounds were found to be non-toxic to both RAW 264.7 and BV2 cells and protected the cells from toxic effects of LPS and IFN- at the higher concentrations. In addition, both compounds had shown similar anti-inflammatory activity in both cell lines at higher concentrations (150 - 250 μM). It was observed that there was no significant difference in anti-inflammatory activities of these compounds. Key words: Vanilloids, RAW264.7 and BV2 cells, Cytotoxicity, Anti- inflammatory activity.

The aim of this study was to synthesise and characterise nanogold-{[(Cu)(phen)(cys)(H2O)]NO3}n conjugate and to evaluate its antiproliferative property against breast cancer cell line (MCF7) and normal cell line (MCF10A). Nanogold solution was prepared using Turkevish method. In one approach, ternary copper(II) complex of 1,10-phenanthroline with amino acid L-cysteine, [(Cu)(phen)(cys)(H2O)]NO3, was first prepared and then tethered with the gold nanoparticles. In another approach, gold nanoparticles were reacted with L-cysteine, copper(II) nitrate, and 1,10-phenanthroline subsequently. The synthesized [(Cu)(phen)(cys)(H2O)]NO3 complex was characterised using fourier transform infrared (FTIR) and electrospray ionisation mass spectrometry (ESI-MS) techniques. FTIR and ESI-MS results show that L-cysteine was bound to the copper through carboxylic and amino groups, with the thiol moiety of L-cysteine remained free. The free thiol group bound to the nanogold surface to complete the nanogold-{[(Cu)(phen)(cys)(H2O)]NO3}n conjugate formation. Nanogold-{[(Cu)(phen)(cys)(H2O)]NO3}n conjugates were characterised using FTIR and UV-Vis spectroscopy. The increase in the surface plasmon absorption band in UV-Vis and absence of thiol peak in FTIR of the conjugate showed that the thiol group of L-cysteine was bound to the gold nanoparticle to form the conjugate. Moreover, the formation of the conjugate is also evidenced from the colour change from ruby red to blue. Anticancer property of the nanogold bound conjugate and unbound analogues was examined using MTS assay on breast cancer cell line (MCF7) and normal cell line (MCF10A). It was concluded that [Cu(phen)(cys)(H2O)]NO3 copper(II) complex was successfully prepared and tethered with nanogold particles. The prepared nanogold-{[(Cu)(phen)(cys)(H2O)]NO3}n conjugate was tested for its antiproliferative activity which showed better antiproliferative property towards the breast cancer cells than the unbound analogues.

Introduction: Photodynamic therapy is an alternative treatment to established standard treatments for cancer and antibiotics for bacterial infections. Although the potential of platinum-based drugs as antibacterial and anticancer agents has been studied for years, due to severe side effects that have limited their further development, ruthenium compounds are now emerging as one of the most promising therapeutic options in terms of targeting cancer and bacterial infections. Objectives: The objective of this work is to prepare and characterize a series of ruthenium complexes, followed by in vitro determination of their antibacterial and anticancer activity. Materials and Methods: Three ruthenium(II) Schiff base {[Ru(bpy)2(SB4CB)]PF6, [Ru(bpy)2(SBFH)]PF6, [Ru(bpy)2(SB4EB)]PF6} and two ruthenium(II) polypyridyl complexes { [Ru(bpy)2(bpy-COOH)](PF6)2, [Ru(bpy)2(bpy-(COOH)2)](PF6)2} were synthesized. FT-IR, UV-Vis, HPLC, NMR, ESI-MS, and elemental analysis were used to characterize the synthesized compounds. The antibacterial activity of the compounds was tested against susceptible strains of gram-negative bacteria Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922 (BSL 1), Acinetobacter baumannii ATCC 19606, and gram-positive Staphylococcus aureus ATCC 35923 (BSL 2). The anticancer activity of the compounds was tested against MDA-MB-231, A549, HT29, MeL28, SW1990 cancer cell lines, and non-cancerous MRC5 cell line. Finally, a combination study of the most promising compound with cisplatin was conducted to determine if there is synergistic anticancer effect. Results: [Ru(bpy)2(SB4CB)]PF6, and [Ru(bpy)2(SBFH)]PF6 were effective against Staphylococcus aureus, with MIC values of 16 μM when tested on irradiated plates and 16 μM and 8 μM, respectively, for non-irradiated plates. In the anticancer assay, SB4EB was active only in the MDA-MB-231 cell line with IC50 of 37.52 μM for light and 39.59 μM for dark. However, [Ru(bpy)2(bpy-COOH)](PF6)2 was inactive towards all the cell lines. The combination of [Ru(bpy)2(SB4CB)]PF6 with cisplatin did not show any synergistic effect on MDA-MB-231 and MeL28 cell lines, but an antagonistic effect was observed for the non-cancerous MRC5 cell line. Conclusion: [Ru(bpy)2(SB4CB)]PF6, and [Ru(bpy)2(SBFH)]PF6 exhibited favorable activity against Staphylococcus aureus. The most promising anticancer results were obtained for [Ru(bpy)2(SB4CB)]PF6 against MDA-MB-231 cell line. However, it’s combination with cisplatin did not show any synergistic effect on MDA-MB-231 and MeL28 cell lines.