Monoamine oxidase inhibitory activities of heterocyclic chalcones / Corné Minders

• Main Author: Minders, Corné
• Language: en
• Published: 2014
• Subjects: Chalcones; Monoamine oxidase inhibitors; Parkinson’s disease; Chalkone; Monoamienoksidase-inhibeerders; Parkinson se siekte
• Online Access: http://hdl.handle.net/10394/12251

Parkinson’s disease is the second most common age-related neurodegenerative disease after Alzheimer’s disease. The characteristic pathological feature of Parkinson’s disease is the loss of neurons in the substantia nigra pars compacta (SNpc), which leads to a striatal dopamine deficiency responsible for the major symptoms of Parkinson’s disease. These symptoms include tremor at rest, postural instability, bradykinesia and in the later stages of Parkinson’s disease, even psychosis. Presently, there is still no cure for Parkinson’s disease and all treatments are only symptomatic. Current research is therefore directed towards the prevention of further dopaminergic neurodegeneration, while the ultimate aim is the reversal of neurodegeneration. Monoamine oxidase (MAO) enzymes are responsible for the regulation and metabolism of monoamine neurotransmitters, such as dopamine. There are two MAO isoforms, MAO-A and MAO-B. Since MAO-B has greater activity in the basal ganglia, it is of particular importance in movement disorders, which include Parkinson’s disease. The selective inhibition of MAO-B, increases dopamine available for binding, and thus reduces Parkinson’s disease symptoms. MAO inhibitors also have neuroprotective potential and thus may slow down, halt and even reverse neurodegeneration in Parkinson’s disease. It is still unclear exactly how MAO inhibitors protect neurons, but one theory suggests that MAO inhibition decreases oxidative stress by reducing the formation of hydrogen peroxide, a metabolic by-product of MAO oxidation of monoamines. Normally, hydrogen peroxide is inactivated by glutathione (GSH), however, in Parkinson’s disease, GSH levels are low, resulting in the accumulation of hydrogen peroxide, which then becomes available for the Fenton reaction. In the Fenton reaction, Fe2+ reacts with hydrogen peroxide and generates an active free radical, the hydroxyl radical. This radical depletes cellular anti-oxidants, damage lipids, proteins and DNA. MAO inhibitors reduce the formation of hydrogen peroxide thus decreasing the formation of hydroxyl radicals and oxidative stress. The MAO inhibitory potential of natural and synthetic chalcones have been illustrated. For example, in 1987, Tanaka and co-workers determined that natural chalcones, such as isoliquiritigenin, have MAO inhibitory activity in rat mitochondria. In 2009, Chimenti and co-workers synthesized a series of 1,3-diphenyl-2-propen-1-ones which exhibited human MAO-B (hMAO-B) selective inhibitory activity. On the other hand, Robinson and co-workers (2013), synthesized novel furanochalcones which also had hMAO-B selective inhibitory activity. A reversible, competitive mode of binding was demonstrated by these compounds. Since the effect of heterocyclic substitution, other than furan on the MAO inhibitory properties of the chalcone scaffold remains unexplored, the aim of this study was to synthesize and evaluate further heterocyclic chalcone analogues as inhibitors of hMAO. RESULTS Design and synthesis: Heterocyclic chalcone analogues that incorporated pyrrole, 5- methylthiophene, 5-chlorothiophene and 2-methoxypyridine substitution were synthesized using the Claisen-Schmidt condensation reaction. All compounds were characterized with 1H-NMR, 13CNMR, IR, MS, and melting points were recorded. Purity was determined with HPLC analysis. MAO inhibition studies: The 50% inhibitory concentration (IC50) values and selectivity index (SI) of all compounds were determined using a fluorometric assay and kynuramine as substrate. Eight out of the ten synthesized compounds exhibited IC50 values < 1 μM, and can thus be considered as potent MAO-B inhibitors, while all compounds showed selectivity for the MAO-B isoform. Compound 10i was the most potent MAO-B inhibitor with an IC50 value of 0.067 μM and the highest SI of 240.7. The most potent MAO-A inhibitor, compound 10f, had an IC50 value of 3.805 μM. Some structure-activity relationships were derived, for example; it was concluded that heterocyclic substitution with 5-methyl-thiophene ring resulted in optimal hMAO-B inhibition, while pyrrole substitution was less favourable. Further investigation is however required as this is only a preliminary study. Reversibility studies: To determine the reversibility of binding, the recovery of enzymatic activity after dilution of the enzyme inhibitor complexes were determined for selected compounds. Results indicated that the most potent MAO-A inhibitor, the pyrrole derivative 10f, had a reversible mode of binding to both the hMAO-B and hMAO-A isoforms, since the enzyme activities were completely recovered by dilution of the inhibitor concentration. In contrast, enzyme activity was only partially recovered after dilution of the most potent MAO-B inhibitor 10i, indicating that this methylthiophene derivative possibly exhibited tight binding to the hMAO-B isoform, and the inhibition caused by this compound was not readily reversed by dilution. In order to determine whether the tight binding as exhibited by compound 10i was due to the thiophene or phenyl moieties, reversibility of binding was also determined for the pyrrole derivative 10e. The results showed that 10e had a reversible mode of binding to the hMAO-B isoform, and enzyme activity was completely recovered by dilution of the inhibitor. Based on these results, it was concluded that the tight binding as exhibited by compound 10i was due to the presence of the thiophene moiety. To confirm that compound 10i exhibited tight, and not irreversible binding, reversibility of binding was also determined by dialysis of enzyme-inhibitor mixtures. For this purpose hMAO-B and 10i, at a concentration of 4 × IC50, were preincubated for a period of 15 min and subsequently dialyzed for 24 h. The results of this study showed that 10i had a reversible mode of binding for MAO-B, since enzyme activity was recovered to a level of 83% after dialysis. Mode of inhibition: To determine the mode of inhibition of compound 10f, Lineweaver-Burk plots were constructed for the inhibition of hMAO-A and hMAO-B. The lines of the Lineweaver-Burk plots intersected at a single point at the y-axis, indicating that 10f had a competitive mode of binding to both hMAO-B and hMAO-A isoforms. MTT viability assay: To determine the toxicity of the chalcones for cultured cells, selected compounds were evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay. The cytotoxicity of the test compounds were evaluated at concentrations of 1 and 10 μM, in HeLa cells. The results indicated that compound 10i was non-toxic at 1 and 10 μM, with 100% and 96% cell viability remaining after 24 h exposure of the compound to the cultured cells. Compound 10f, however, exhibited significant toxicity at 10 μM, with only 5% viable cells remaining. In contrast, compound 10e, with the same pyrrole moiety as 10f, was non-toxic at 1 μM and 10 μM, with 99% and 98%, cell viability remaining. It was concluded that the pyrrole moiety of 10f was not responsible for its higher degree of cytotoxicity, which suggests that the CF3 substituent may play a role in the higher degree of cytotoxicity observed for 10f. Further investigation is required to determine the mode of cytotoxicity, when cultured cells are exposed to 10f. Docking Studies: To complete this study and rationalise the results of the MAO inhibition studies, molecular modelling was carried out and all compounds were docked into the crystal structure of hMAO-B, by using the CDOCKER module of Discovery Studio. Some insights were obtained regarding the binding of compound 10i. This compound bound to MAO-B with the phenyl ring facing the FAD cofactor. This orientation allowed for the formation of pi-pi interaction with Tyr 398 as well as a pi-sigma interaction between the thiophene ring and Ile 199 (which is part of the gating switch of MAO-B). It is speculated that the tight binding component of hMAO-B inhibition by 10i may, at least in part, be attributed to the interaction of this compound with the gating switch amino acid, Ile 199. The docking results also showed that most compounds interacted with Tyr 326 or Tyr 398, while interactions with Cys 172, Gln 206, Ile 199 and Tyr 435 also occurred. In conclusion, novel heterocyclic chalcone analogues with promising MAO-B inhibitory activities were successfully synthesized and evaluated. === MSc (Pharmaceutical Chemistry) North-West University, Potchefstroom Campus, 2014