| dc.description.abstract | Monoamine oxidase (MAO) consists of two isoforms, MAO-A and MAO-B. MAO is responsible for the oxidation of neurotransmitter and dietary amines. The MAO-B isoform, in particular, is considered to be a drug target for the treatment of
neurodegenerative disorders such as Parkinson’s disease (PD). Inhibition of MAO-B
may conserve dopamine in the brain, resulting in symptomatic relief of PD. Furthermore, inhibition of MAO-B may prevent the formation of neurotoxic metabolic products and thus MAO-B inhibitors may exert a neuroprotective effect. For these reasons, MAO-B inhibitors have been used as antiparkinsonian agents. The MAO-B inhibitors that are currently being used in the treatment of PD are irreversible inhibitors. In addition to the potential adverse effects associated with irreversible inhibition, the recovery of MAO activity after inactivation by an irreversible inhibitor may require several weeks. Reversible inhibitors may have less adverse effects and enzyme recovery after withdrawal is almost immediate. In this study three series of novel reversible MAO inhibitors were synthesized and their MAO inhibitory properties were examined. For the first two series, caffeine was employed as a scaffold with alkyloxy substitution on C8 of the caffeine moiety. Caffeine is a weak inhibitor of MAO-B, but substitution on C8 increases its inhibitory activity towards MAO-B as illustrated with the potent MAO-B inhibitor, (E)-8-(3-chlorostyryl)caffeine (CSC). In a previous study it was demonstrated with 8-benzyloxycaffeine that an alkyloxy side chain may result in potent inhibitors of both
MAO-A and –B. Based on these reports the first two series of caffeine analogues contained alkyloxy side chains on C8 of the caffeine. The target caffeine analogues were synthesized by condensing an appropriately substituted alcohol with 8-chlorocaffeine. For the third series, 6-benzyloxyphthalide was used as lead compound and a series of phthalide analogues were synthesized with various alkyloxy side chains on the C6 position. In previous studies it has been shown that benzyloxy substitution on the C5
positions of isatin and phthalimide resulted in potent MAO inhibitors. Isatin and
phthalimide are structurally related to phthalide and the C5 position on isatin and
phthalimide are homologues to the C6 position on phthalide. Therefore, the third
series explored the MAO inhibitory properties of phthalide analogues with alkyloxy side chains on C6. To investigate the importance of the oxy moiety for MAO inhibitory activity, the effects of benzylamino substitution on the C6 position of the phthalide ring was also examined. The alkyloxyphthalide analogues were synthesized by reacting 6-hydroxyphthalide with an appropriately substituted alkyl bromide. The benzylaminophthalide was synthesized, in turn, according to the same procedure from 6-aminophthalide and benzyl bromide. The synthesized compounds were evaluated as inhibitors of recombinant human MAO-A and –B. Kynuramine, a MAO-A/B mixed substrate served as enzyme
substrate. Kynuramine is oxidized by the MAOs to yield 6-hydroxyquinoline, a compound which fluoresces in alkaline media. Concentration measurements of 6-
hydroxyquinoline can conveniently be made via fluorescence spectrophotometry since both kynuramine and the inhibitors are non-fluorescent under these assay conditions. The inhibition potencies of the test compounds were expressed as the
corresponding IC50 values. Representative inhibitors in each series were selected to
evaluate the reversibility of inhibition of the compounds. In the first series, the 8-aryl and alkyloxycaffeine analogues were found to be
reversible inhibitors of MAO-A and –B with greater selectivity towards MAO-B. A
particularly potent MAO inhibitor among the first series was 8-[2-(4-
bromophenoxy)ethoxy]caffeine with IC50 values of 1.65 μM and 0.166 μM towards
MAO-A and -B, respectively. Based on the promising inhibitory activities of the first
series, in the second series, the MAO inhibitory properties of the 8-(2-
phenoxyethoxy)caffeine analogues were further explored. For this purpose, 8-(2-
phenoxyethoxy)caffeine analogues with different substituents on C4 of the phenyl
ring were synthesized. Structure-activity relationship (SAR) studies were carried out
in order to correlate selected physicochemical properties of the C4 substituents with the inhibitory activities towards MAO. It was found that substituents on C4 of the phenyl ring which are more lipophilic, electron withdrawing and has greater bulkiness may result in more potent inhibition towards MAO-A. The most potent MAO-A inhibitor of this series was 8-[2-(4-iodophenoxy)ethoxy]caffeine with an IC50 value of 0.924 μM. The results also documented that MAO-B inhibition potency correlated with the electronic parameters of the substituent on C4 of the phenyl ring, with highly electron withdrawing substituents yielding potent MAO-B inhibitors. This behaviour is exemplified by 8-[2-(4-trifluoromethylphenoxy)ethoxy]caffeine, which inhibited MAO-B with an IC50 value of 0.061 μM. In the third series the phthalide analogues also proved to be potent reversible inhibitors of both MAO-A and –B. As observed with the caffeine derived compounds of series 1 and 2, these compounds were also more selective towards MAO-B. The most potent MAO-B inhibitor was 6-[4-
(trifluoromethyl)benzyloxy]phthalide with an IC50 value of 0.0014 μM while the most
potent MAO-A inhibitor was 6-(3-phenylpropoxy)phthalide with an IC50 value of 0.1 μM. The least potent inhibitor of MAO-A and –B was 6-(benzylamino)phthalide which inhibited MAO-A with an IC50 value of 59.9 μM and displayed no inhibition towards
MAO-B. To examine the reversibility of the target compounds, two methods were employed. For the first two series, the caffeine derived inhibitors, 8-[2-(4-
bromophenoxy)ethoxy]caffeine was selected as representative inhibitor. MAO-A and
–B were pre-incubated with the representative inhibitor for time periods of 0, 15, 30 and 60 minutes and the residual catalytic rates were measured. The results showed that there was no significant time-dependent decrease in catalytic rate, which is an indication that the representative inhibitor is a reversible inhibitor of MAO-A and –B. For the third series, reversibility of MAO-A and –B inhibition was determined by using 6-(3-phenylpropoxy)phthalide and 6-[4-(trifluoromethyl)benzyloxy]phthalide as
representative inhibitors, respectively. MAO was pre-incubated with the respective
inhibitors at concentrations of 10-fold IC50 and 100-fold IC50. After dilution of the
incubations to 0.1-fold IC50 and 1-fold IC50, the residual enzyme activities were measured. After the dilution to 1-fold IC50, the activities of MAO-A and –B were
recovered to 86% and 68%, respectively. This behavior is consistent with reversible
inhibition. To further establish the reversibility of inhibition by the representative caffeine and phthalide inhibitors, Lineweaver-Burk plots were constructed. The results showed that the Lineweaver-Burk plots intersected on the y-axis, which is an indication that the representative inhibitors are competitive and therefore reversible inhibitors of MAO-A and –B. Reversible inhibition is in general a desirable trait since irreversible MAO inhibitors are frequently associated with unfavourable and potentially fatal side effects. Based on the findings that the caffeine and phthalide analogues examined in this study are potent and reversible inhibitors of MAO-A and –B, these compounds represent suitable candidates for the development of novel MAO inhibitors for the treatment of PD. | en_US |
