Faculty of Applied Sciences
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Item Synthesis of novel quinoline derivatives and their cytotoxicity in A549 lung cancer cells(2017) Nkosi, S'busiso Mfan'vele; Gengan, Robert Moonsamy; Chuturgoon, Anil A.Quinoline and its derivatives represent an important class of nitrogen-containing heterocylces as they are useful intermediates in organic synthesis and possess a broad spectrum of biological activities, such as anti-asthmatic, anti-inflammatory and anti-malarial activity. Hence, synthesis of novel compounds with potent biological activities is important in medicine. Significant research is directed into the development of new quinoline based structures and new methods for their preparations. In the past, synthesis of complex molecules was accomplished by step-wise reaction. This was time consuming and yield was generally low. Nowadays, multi-component reactions (MCRs) are being used since three or more substrates can be reacted in a one-pot reaction. Therefore yields are higher and the reaction is more efficient. In this research investigation novel quinoline derivatives, using the multi-component reaction protocol, were synthesized. After characterization of the product by several spectroscopic techniques, the biological potential of these compounds were assessed using lung cancer cell lines, bacteria and molecular modeling in an enzymatic system. In the synthetic part of this study, the first step was the preparation of the starting compound 2- chloro-3-formyl quinoline for which the Vilsmeier-Haack cyclisation protocol was used. The cyclisation was carried out by combining DMF and POCl3 at 5°C to form an electrophile which then reacted in situ with N-phenylacetamide at 100ºC to afford 2-chloro-3-formyl quinoline in high yield (95%). This was followed by the synthesis of a series of novel quinoline derivatives in a MCR system comprising 2- chloro-3-formyl quinoline, malononitrile, aromatic amines and dimethyl acetylenedicarboxylate in the presence of a catalytic amount of triethylamine. Valuable features of this routine included high yields, extensive substrate range and straight forward procedures. Eight novel poly-functionalised dihydropyridine quinoline derivatives were synthesized, purified and characterized. The outline for the synthesis of poly-functionalised dihydropyridine quinoline derivatives is presented graphically in Scheme 1. Scheme 2 shows the eight compounds synthesized and used subsequently for further studies. Step 1 CH3 a N O H CHO N Cl Step 2 CHO CN N Cl CN NH2 R O OCH3 b OCH3 O MeO2C MeO2C N Cl CN N NH2 R = m-CH3, o-OCH3, p-Cl, m,p-Cl, o-F, m-F, p-F R Reaction Conditions: a. DMF, POCl3 b. Et3N, EtOH Scheme 1: Graphical representation for the synthesis of poly-functionalised dihydropyridine quinoline derivatives The novel eight compounds were screened for their potential activity in lung cancer cell lines. A549 cells were incubated for 24 hours with a range of concentrations of each compound, in triplicate, in a micro-titre plate together with an untreated control. Each experiment was conducted twice on separate occasions; the results from the first set matched the repeated experiment. The cells were then incubated (37ºC, 5% CO2) with the MTT substrate for 4 hours. Thereafter all supernatants were aspirated and DMSO was added to the wells. Finally the optical density was measured at 570 nm at a reference wavelength of 690 nm with an ELISA plate reader. The net MTT dependant absorbance (optical density) of each sample was calculated by subtracting the average absorbance of the blank from the average absorbance of each sample. Data were represented as mean optical density plus or minus the standard deviation. Four of the synthesized compounds (A1-A8) were evaluated for their cytotoxicity activities. The anti-cancer assay indicated that poly-functionalised dihydropyridine quinoline compounds, A2, A3 and A4 have good potential as anti-cancer drugs. Among them, A2 and A4 proved to be dose dependent with A4 having the highest toxicity at 250 µM and A8 having the highest toxicity at 125, 250 and 500 µM, whereas A1, A5, A6 and A7 were not cytotoxic. O H3CO H3CO O N Cl CN NH2 O H3CO H3CO O N Cl CN N NH2 OCH3 O H3CO H3CO O N Cl CN N NH2 O H3CO H3CO O N Cl CN NH2 CH3 Cl A1 A2 A3 A4 O H3CO H3CO O N Cl CN N NH2 F O H3CO H3CO O N Cl CN N NH2 O H3CO H3CO O N Cl CN NH2 O H3CO H3CO O N Cl CN N NH2 F Cl F Cl A5 A6 A7 A8 Scheme 2: Structures of novel poly-functionalised dihydropyridine quinoline derivatives by MCRs Since molecular docking is a key tool in structural molecular biology and computer-assisted drug design, these compounds were subjected to molecular docking and the binding mode for the compounds, within the active site of the protein, was analyzed. Docking of A1 to Human mdm2 protein provided insights into the binding regions. Three hydrogen bonds were formed between GLU 25 (2.7 Å distance), LEU 27 (3.2 Å distance) and LEU 54 (3.2 Å distance) atoms with binding energy of -8.91 kcal/mol. Docking of A1 with Human mdm2 indicated the lowest binding energy thereby showing strong affinity of the ligand molecule with the receptor which has been stabilized by strong hydrogen bond interactions in the binding pocket. This confirms that A1 is a better inhibitor for E3 ubiquitin-protein ligase mdm2 than all the other compounds tested (A2-A8). Further, the eight novel poly-functionalised dihydropyridine quinoline derivatives were evaluated for their antibacterial activity. This was performed using the MABA method against three strains i.e. Gram negative; Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 25922) and Gram positive; Staphylococcus aureus (ATCC 29213) using the broth micro dilution method. Standard antibiotics (ciprofloxacin and nalidixic acid) were used as positive controls and DMSO was used as a negative control. The results obtained from the anti-bacterial assay showed that compounds A4, A7 and A8 have high activity, whereas A2 and A3 showed poor activity against all the tested bacterial strains. Compound A6 showed no activity against S. aureus and E. coli.Item Pharmacological Screening of Substituted 1, 4 Dihydropyrimidines(2016) Govender, Reshme; Odhav, Bharti; Venugopala, Katharigatta NarayanaswamyPharmacological 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.