The synthesis and evaluation of (E)-styrylisatin analogues as inhibitors of monoamine oxidase B

Abstract

Monoamine oxidase B (MAO-B) inhibitors are currently clinically used in the symptomatic treatment of Parkinson's disease (PO) and may also possess .neuroprotective activity. The irreversible MAO-'B inhibitor, (R)-deprenyl, .is commonly used in PO treatment, usually in combination with levodopa during dopamine replacement therapy. In contrast with reversible inhibition, .enzyme recovery after irreversible inhibition involves de novo synthesis. The relatively quick return of enzyme activity makes potent reversible MAO-B inhibitors safer and more desirable. Both isatin and caffeine are small molecules that have been reported to inhibit MAO-B. !satin is a relatively good endogenous inhibitor (K = 3 µM) whereas caffeine is a weak inhibitor (K1 = 650 µm) . The inhibitory potency of caffeine have been improved by substitution at C-8 of the caffeine ring with a styryl side-chain. Addition of an electron withdrawing substituent at C-3 of the phenyl ring produced structures with exceptional reversible MAO-B .inhibitory potency, for example (E)-8-(3-chlorostyryl)caffeine (CSC) (K1 = 0.1 µM) .In this study we investigated whether styryl substitution of the lead compound, isatin, at C-5 and C-6 will similarly enhance isatin's MAO-B inhibitory potency. Preliminary computer modelling of the proposed (E)-5-sty.rylisatin and (E)-6-styrylisatin analogues, as well as isatin, into the active site of recombinant MAO-B supported the hypothesis of increased MAO-B inhibitory activity for these styryl analogues. The styryl sidechain seems to be stabilised in the entrance cavity of the enzyme during binding, while the isatin moiety is located in the substrate-binding cavity where it is involved in hydrogen bonding. This duel binding mode is similar to that proposed for the styrylcaffeines and is thought to facilitate the potent MAO-B inhibition of styrylcaffeines. After successful synthesis of the (E)-5-styrylisatin and (E)-6-styrylisatin analogues, the compounds were evaluated in vitro as reversible inhibitors of baboon liver MAO-B. Inhibitory activity of the styrylisatin analogues were determined with a spectrophotometric method. The inhibitory potencies for all the styrylisatin analogues were expressed in terms of the concentration of the compound necessary for 50% inhibition of the enzyme in vitro (ICsa value). Reversibility of inhibition was confirmed with a time-dependent inhibition study that showed that the potencies of inhibition of MAO-B by the (E)-styrylisatin analogues were independent of the time period for which the analogue was incubated with the enzyme. The results confirmed that substitution with a styryl side-chain on C-5 of the isatin ring resulted in reversible inhibitors of MAO-B that are exceptionally potent. For example, (E)-5-

(3-chlorostyryl)isatin (IC50 = 20.7 nM) was found to be 430 times more potent than the lead ,compound isatin (IC50 = 8.6 µM). Also, (E)-5-styrylisatin was found to have a IC50 value of 41.7 nM, which is approximately 210 times more potent as a MAO-B inhibitor than isatin. The (E)-6-styrylisatin analogues showed moderate, reversible MAO-B inhibition. For example, (E)-6-styrylisatin has an IC5o value of 436.8 nM. The styryl side-chain as well as electronic and lipophilic properties seem to be important for potent inhibition of MAO-B by these compounds. In this study, the synthesised (E)-5-styry\isatin analogues, in particular (E)-5-(3- chlorostyryl)isatin, showed potential as novel reversible MAO-B inhibitors. Synthesis and evaluatin of a more extensive series of styrylisatin analogues would enable a Hansch-type structure-activity relationship study that could identify the optimal physicochemical properties for the compounds.