Submitted in partial fulfillment for the Degree of Master of Applied Sciences in Biotechnology, Durban University of Technology, Durban, South Africa, 2016. / Pharmacological research is essential for the advancement of treatment therapies to combat diseases that plague mankind. Pyrimidines have been a subject under investigation by medicinal chemists for many years due to their interesting pharmacological properties. In previous studies, pyrimidines and their derivatives have been reported to have antimicrobial, anti-inflammatory, antimalarial, analgesic, and antitumour activities amongst other biological activities. Although there has been a significant amount of research carried out on these heterocycles, there will always be a continuous need for the discovery of novel synthetic drugs which have a higher degree of potency and fewer side effects. Hence, this study was undertaken to determine the pharmacological activities of eight novel 1, 4 dihydropyrimidine analogues (DHPM 1 – 8), that have been synthesized in our laboratory. The dihydropyrimidines were synthesized and characterized and thereafter evaluated for in vitro antimicrobial, antioxidant, anti-inflammatory, cytotoxicity and apoptotic activities. The compounds also underwent a safety study. Antimicrobial activity was evaluated using the disk diffusion assay; compounds displaying superior activity were subjected to further analysis to establish the minimum inhibitory concentration. Overall compounds DHPM 7 and 8 showed the best antibacterial activity against Gram positive bacteria. The minimum inhibitory concentration (MIC) for DHPM 7 against the Gram positive organisms (B.cereus, S.aureus and B.coagulans) was 0.75 µg/mL; however DHPM 7 had a MIC of 0.37 µg/mL against M. luteus. DHPM 8 displayed an MIC of 0.75 µg/mL against B.cereus, S.aureus, M.luteus, S.faecalis and B.coagulans. Antioxidant activity was assessed using the DPPH method. DHPM 2 showed outstanding free radical scavenging capacity of 90.63% at a concentration of 1 mg/mL. The DHPM 1 - 8 were analysed for their lipoxygenase inhibitory activity. Excellent inhibition ranging from 59.37 ± 0.6 to 81.19 ± 0.94% was demonstrated. The inhibitory activity was elucidated by a molecular docking study against the lipoxygenase enzyme (PDB code = 3V99) using the MOE 2013.08 and Leadit 2.1.2 software and high affinities were demonstrated. DHPM 1 - 8 were tested for cytotoxic activity against two human cancer cell lines, MCF-7 and UACC-62 by means of the MTT assay. It was observed for the MCF-7 cell line, DHPM 1, 4, 6, 7 and 8 displayed cytotoxicity above 89% at 50 µg/mL. The DHPMs at 50 µg/mL were noted to be very effective against the Melanoma cell line with DHPM 2 having a cytotoxicity value of 82.62% and DHPM 1, 4, 5, 6, 7 and 8 exhibiting cytotoxicity greater than 96%. Only slight inhibition of the proliferation of PBMC’s was noted. IC50 values of DHPM 1-8 were determined and the best activity overall was displayed by DHPM 8. The IC50 of DHPM 8 was 0.92 ± 0.09 and 1.97 ± 0.08 µM against MCF - 7 and UACC - 62 cell lines, respectively. The compounds that displayed toxicity towards the UACC - 62 cell line were investigated for their apoptotic inducing potential. The apoptotic studies were performed by flow cytometry using the following assays; Annexin V, JC-1 and Caspase -3 assays. The effect of these compounds was compared to a known anti-cancer drug, Camptothecin. On evaluation of the mechanism of action of the compounds, it was found that most compounds are using apoptotic pathways for cell death. Our studies have identified antimicrobial activity (DHPM 1-8) against Gram positive organisms, high antioxidant activity (DHPM 2), anti-inflammatory activity (DHPM 1-8) and anticancer activity (DHPM 1-8) against UACC-62 and MCF-7 cells. DHPM 1-8 were found to have no toxicity at 100 µg/mL in the brine shrimp assay and hence are probably safe as therapeutic agents. Furthermore molecular docking studies confirmed the activity of DHPM 1-8 as potential lipoxygenase inhibitors. DHPM 1-8 are novel compounds with great potential to be developed into chemotherapeutic agents. / M

One current approach in the treatment of cancer is the inhibition of cyclin dependent kinase (CDK) enzymes with small molecules. Here the discovery and development of 2-anilino-4-(thiazol-5-yl)pyrimidine CDK inhibitors is described, including details of the design and successful synthesis of novel ring-constrained thiazolylpyrimidines. The structure-activity relationship (SAR) trends exhibited by this constrained thiazolylpyrimidine family of CDK inhibitors are presented and compared with those from an unconstrained series of analogues. One significant finding from this aspect of the project was that ring-constrained thiazolylpyrimidines in general inhibit CDK2-cyclin E with greater potency than the corresponding unconstrained forms. Furthermore, an X-ray crystal structure of 2-methyl-N-[3-nitrophenyl]-4,5-dihydrothiazolo[4,5-h]quinazolin-8-amine, a representative from the constrained thiazolylpyrimidine series, in complex with CDK2-cyclin A is reported; confirming the binding mode within the CDK2 ATP binding pocket. A further assessment of SARs through the synthesis of control compounds and an extended study into the synthesis of N-substituted derivatives is described. The identification of CDK inhibitors that possess a strong selectivity profile across the CDK family is important. For example, the identification of highly CDK4-selective inhibitors should enable researchers to study the biological role of this important enzyme and to enable a block of cell division in the G1 phase. Here synthetic attempts to prepare a potentially CDK4 selective inhibitor compound, namely 5-methyl-N8-[4-(piperazin-1-yl)phenyl]thiazolo[4,5-h]quinazoline-2,8-diamine, are described. This approach was inspired by SAR data published on a structurally related inhibitor, 8-cyclopentyl-5-methyl-2-[4-(piperazin-1-yl)phenylamino]pyrido[2,3-d]pyrimidin-7(8H)-one.

The importance of metabolism in tumor initiation and progression is becoming increasingly clear. Metabolic changes induced by oncogenic drivers of cancer contribute to tumor growth and are attractive targets for cancer treatment. Phosphatase and Tensin homolog deleted from chromosome ten (PTEN) is one of the most commonly mutated tumor suppressors in cancer and operates in multiple roles, rendering it a hub for understanding cancer biology and for developing targeted therapy. PTEN’s canonical function is its ability to antagonize the phosphoinositide 3-kinase (PI3K) pathway by dephosphorylating the lipid second messenger phosphatidylinositol (3,4,5) tri-phosphate (PIP3). This thesis focuses on the effects of PTEN loss on cellular metabolism, and the therapeutic vulnerability that stems from metabolic alterations. First, we discovered that loss of Pten in mouse embryonic fibroblasts (MEFs) increases cellular proliferation and the number of replication forks per cell, launching our investigation into metabolic pathways that may be altered to support increased growth. Indeed, we found that Pten-/- cells exhibited a dependence on glutamine for their faster rate of growth, and that glutamine was channeled into the de novo synthesis of pyrimidines. The next chapter examined dihydroorotate dehydrogenase (DHODH), a rate limiting enzyme for pyrimidine ring synthesis in the de novo pyrimidine synthesis pathway. We found that PTEN-deficient primary cells and cancer cell lines were more sensitive to inhibition of DHODH than PTEN WT cells were, and that the growth inhibition could be rescued by metabolites downstream of DHODH. Furthermore, we found that xenografted human triple negative breast cancer tumors in mice could be diminished by treatment with leflunomide, a DHODH inhibitor. In the following chapter, we aimed to identify the mechanisms leading to cell death in PTEN mutant cells upon DHODH inhibition. We found that inherent defects in checkpoint regulation in PTEN-deficient cells were exacerbated by the stress of obstructed de novo pyrimidine synthesis, leading to a buildup of DNA damage at replication forks and ultimately chromosomal breaks. This was instigated by AKT-mediated phosphorylation of TOPBP1 that caused inadequate ATR activation, as well as AKT-mediated phosphorylation and inactivation of CHK1. In sum, the findings of this thesis indicate that enhanced glutamine flux to de novo pyrimidine synthesis in PTEN mutant cells generates vulnerability to DHODH inhibition. The integration of altered glutamine regulation with PTEN’s effect on replication, DNA damage, and the checkpoint response manifests as synthetic lethality upon DHODH inhibition in cells with PTEN inactivation. Inhibition of DHODH could thus be a promising therapy for patients with PTEN mutant cancers.