Parkinson’s disease (PD) is an age-related neurodegenerative disorder. The degeneration of the neurons of the substantia nigra in the midbrain leads to the loss of dopamine from the striatum, which is responsible for the motor symptoms of PD. In the brain, the enzyme, monoamine oxidase B (MAOB), An analysis of the Lineweaver-Burk plots indicated that 7-(3-bromobenzyloxy)-3,4-dihydro-2(1H)- quinolinone inhibits MAO-B with a Ki value of 2.7 nM. An analysis of the structure-activity relationships for MAO-B inhibition shows that substitution on the C7 position of the 3,4-dihydro- 2(1H)-quinolinone moiety leads to significantly more potent inhibition compared to substitution on C6. In this regard, a benzyloxy substituent on C7 is more favourable than phenylethoxy and phenylpropoxy substitution on this position. In spite of this, C6-substituted 3,4-dihydro-2(1H)-quinolinone with potent MAO-B inhibitory activities were also identified. An analyses of selected properties of the 3,4-dihydro-2(1H)- quinolinones showed that the compounds are highly lipophilic with logP values in the range of 3.03- 4.55. LogP values between 1 and 3 are, however, in the ideal range for bioavailability. The compounds synthesised have logP values higher than 3, which may lead to lower bioavailability. Laboratory data further showed that none of the 3,4-dihydro-2(1H)-quinolinones are highly toxic to cultured cells at the concentrations, 1 μM and 10 μM, tested. For example, the most potent MAO-B inhibitor, 7-(3-bromobenzyloxy)-3,4-dihydro-2(1H)-quinolinone, reduced cell viability to 88.11% and 86.10% at concentrations of 1 μM and 10 μM, respectively. These concentrations are well above its IC50 value for the inhibition of MAO-B. At concentrations required for MAO-B inhibition, the more potent 3,4-dihydro-2(1H)-quinolinones are thus unlikely to be cytotoxic. It may thus be concluded that C7-substituted 3,4-dihydro-2(1H)-quinolinones are promising highly potent and selective MAO-B inhibitors, and thus leads for the therapy of Parkinson’s disease. represents a major catabolic pathway of dopamine. Inhibitors of MAO-B conserve the depleted supply of dopamine and are thus used in the therapy of PD. In the present study, a series of 3,4- dihydro-2(1H)-quinolinone derivatives were synthesized and evaluated as inhibitors of recombinant human MAO-A and MAO-B. These quinolinone derivatives are structurally related to a series of coumarin (1-benzopyran-2-one) derivatives, which has been reported to act as MAO-B inhibitors. C6- and C7-substituted 3,4-dihydro-2(1H)-quinolinone derivatives were synthesized by reacting 6- or 7- hydroxy-3,4-dihydro-2(1H)-quinolinone with an appropriately substituted alkyl bromide in the presence of base. To evaluate the MAO inhibitory properties (IC50 values) of the quinolinone derivatives the recombinant human MAO-A and MAO-B enzymes were used. The reversibility of inhibition of a representative 3,4-dihydro-2(1H)-quinolinone derivative was examined by measuring the recovery of enzyme activity after the dilution of the enzyme-inhibitor complexes, while the mode of MAO inhibition was determined by constructing Lineweaver-Burk plots. To determine the lipophilicity of the 3,4-dihydro-2(1H)-quinolinone derivatives, the logP values were measured. The toxicity of the 3,4-dihydro-2(1H)-quinolinone derivatives towards cultured cells (cytotoxicity) was also measured. The results document that the 3,4-dihydro-2(1H)-quinolinone derivatives are highly potent and selective MAO-B inhibitors with most homologues exhibiting IC50 values in the nanomolar range. The most potent MAO-B inhibitor, 7-(3-bromobenzyloxy)-3,4-dihydro-2(1H)-quinolinone, exhibits an IC50 value of 2.9 nM with a 2750-fold selectivity for MAO-B over the MAO-A isoform. As a MAO-B inhibitor, this compound is approximately equipotent to the most potent coumarin derivative (IC50 = 1.14 nM) reported in literature. Since MAO-B activity could be recovered after dilution of enzyme-inhibitor mixtures, it may be concluded that 7-(3-bromobenzyloxy)-3,4-dihydro-2(1H)- quinolinone is a reversible MAO-B inhibitor. The Lineweaver-Burk plots constructed for the inhibition of MAO-B by 7-(3-bromobenzyloxy)-3,4-dihydro-2(1H)-quinolinone were linear and intersected on the y-axis. These data indicated that this compound also is a competitive MAO-B inhibitor. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014

Very little is known about why and how the Parkinson's disease (PD) neurodegenerative process begins and progresses. In the course of developments for treatment of PD, the discovery of the inhibition of monoamine oxidase (MAO B) was a conceptual breakthrough, and has now been firmly established. MAO B has also been implicated in the neurodegenerative processes resulting from exposure to xenobiotic amines. For example, MAO B catalyzes the first step of the bioactivation of the parkinsonian inducing pro-neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Additional insight into the mechanism of catalysis of MAO B and the mechanism of neurotoxicity by MPTP is therefore very valuable in the pursuit of the treatment of PD. / Thesis (Ph.D. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2008.

Parkinson’s disease (PD) is a neurodegenerative disorder, which is progressive in nature and usually associated with the elderly. It is the second most common age-related neurodegenerative disorder after Alzheimer’s disease (AD). PD occurs when there is a dramatic loss of dopamine (DA) in the striatum, a substructure of the basal ganglia, of the brain due to the degeneration of the nigrostriatal pathway that contains the dopaminergic neurons. Motor symptoms of PD include bradykinesia, muscular rigidity and resting tremors. Non-motor symptoms include speech and sleep problems, hallucinations and depression. Diverse treatment options are available to treat the symptoms of PD, including levodopa (L-Dopa), DA agonists and monoamine oxidase B (MAO-B) inhibitors. The MAOs are flavoproteins that are bound to the outer membrane of the mitochondria and catalyze the oxidative deamination of neurotransmitters such as serotonin (5-HT), noradrenaline (NA) and DA. Two isoforms occur, namely MAO-A and –B, which share a 70% sequence identity. MAO-A catalyzes the oxidation of 5-HT and MAO-B has a substrate specificity towards benzylamine and 2-phenylethylamine. DA, NA, adrenaline and tryptamine are oxidized by both forms. MAO-A plays an important role in depression while MAO-B plays an important role in PD. The two isoforms are not evenly distributed in the brain. Of particular relevance to PD is the observation that, in the basal ganglia, MAO-B is the predominant form and the oxidation of DA in this region is largely due to MAO-B activity. Also, with an increase in age, there is an up to fourfold increase in MAO-B activity in the brain. In the aged parkinsonian brain, MAO-B is therefore a major DA metabolizing enzyme and MAO-B inhibitors have an important role in the therapy of PD. MAO-B inhibitors may potentially reduce the metabolic destruction of DA and thereby provide relief from the symptoms of PD. MAO-B inhibitors may also exert a neuroprotective effect in PD. In the catalytic cycle of MAO-B, one mole each of an aldehyde, hydrogen peroxide and ammonia are formed for each mole of primary amine substrate oxidized. Ferrous iron, which is abundant in the basal ganglia, may react with the hydrogen peroxide to form hydroxyl radicals in the Fenton reaction. The hydroxyl radical damages virtually all types of biomolecules including proteins, DNA, lipids, carbohydrates and amino acids. The aldehyde, in turn, may react with amino groups of proteins, and thus lead to cell injury. Inhibitors of MAO-B may reduce the MAO-catalyzed formation of hydrogen peroxide and aldehydes in the basal ganglia, and thus act as neuroprotective agents. MAO-B inhibitors that are currently being used in the treatment of PD are selegiline and rasagiline. Both are irreversible inhibitors of MAO-B. While irreversible inhibitors of MAO have been used extensively as drugs, irreversible inhibition has a number of disadvantages. These include the loss of selectivity as a result of repeated drug administration and a slow and variable rate of enzyme recovery following termination of drug treatment. The turnover rate for the biosynthesis of MAO-B in the human brain may require as much as 40 days while with reversible inhibition, enzyme activity is recovered when the inhibitor is eliminated from the tissues. For these reasons the discovery of novel MAO-B inhibitors, which interact reversibly with the enzymes are of value in the therapy of PD. The goal of this study was to design novel and reversible inhibitors of MAO-B, which may find application in the therapy of PD. In the current study, caffeine was used as scaffold for the design of new MAO inhibitors. Caffeine is reported to be a weak inhibitor of MAO-B, with an IC50 value of 5084 μM. Substitution at C-8 of the caffeine moiety, however, yields compounds with potent MAO-B inhibitory properties. Of particular importance to this study is a recent report that a series of 8-sulfanylcaffeine analogues acts as selective inhibitors of human MAO-B. Among the compounds examined, 8-[(phenylethyl)sulfanyl]caffeine was found to be a particularly potent MAO-B inhibitor with an IC50 value of 0.223 μM. In an attempt to further enhance the MAO-B inhibition potency of 8-[(phenylethyl)sulfanyl]caffeine, and possibly to discover highly potent MAO-B inhibitors, a series of five 8-[(phenylethyl)sulfanyl]caffeine analogues was synthesized and evaluated as inhibitors of human MAO-A and –B. For the purpose of this study 8- [(phenylethyl)sulfanyl]caffeine homologues containing C-3 alkyl (CF3, CH3 and OCH3) and halogen (Cl and Br) substituents on the phenyl ring were considered. Furthermore, a series of two 8-sulfinylcaffeine analogues and one 8-sulfonylcaffeine were synthesized and their MAO inhibitory potencies were measured. The purpose with these compounds was to compare the MAO inhibitory properties of the 8-sulfinylcaffeine analogues and 8-sulfonylcaffeine with those of the 8-sulfanylcaffeine analogues. This study also investigates the MAO inhibition properties of three selected 8-[(phenylpropyl)sulfanyl]caffeine and two 8-(benzylsulfanyl)caffeine analogues. Chemistry: The target 8-sulfanylcaffeine analogues were synthesized according to the literature procedure. 8-Chlorocaffeine was reacted with an appropriate mercaptan in the presence of NaOH, to yield the target 8-sulfanylcaffeine analogues in yields of 6.4–50.7%. 8-Chlorocaffeine, in turn, was conveniently synthesized in high yield by reacting chlorine with caffeine in chloroform. In certain instances, the mercaptan starting materials were not commercially available and were thus synthesized according to the literature procedure by reacting an appropriate alkylbromide with thiourea. The resulting thiouronium salt was hydrolyzed in the presence of NaOH to yield the target mercaptan. The 8-sulfinylcaffeine analogues and 8- sulfonylcaffeine were synthesized by reacting the 8-sulfanylcaffeines with H2O2 in the presence of glacial acetic acid and acetic anhydride. The structures and the purities of the inhibitors were verified by NMR, MS and HPLC analyses. MAO inhibition studies: The MAO inhibitory properties of the test compounds were examined using the recombinant human enzymes. The mixed MAO-A/B substrate, kynuramine, was employed as substrate for both enzymes and the inhibition potencies were expressed as the IC50 values. The 8-[(phenylethyl)sulfanyl]caffeine analogues were found to be highly potent inhibitors of MAO-B. The IC50 values recorded for these homologues ranged from 0.017–0.125 μM, making them twofold to 13-fold more potent MAO-B inhibitors than the lead compound, 8- [(phenylethyl)sulfanyl]caffeine (IC50 = 0.223 μM). For comparison, the reversible MAO-B selective inhibitor, lazabemide, exhibits an IC50 value of 0.091 μM under the same conditions (unpublished data from our laboratory). Interestingly, both alkyl (CF3, CH3 and OCH3) and halogen (Cl and Br) substitution lead to highly potent MAO-B inhibition. It may therefore be concluded that substitution on C-3 is a general strategy to enhance the MAO-B inhibition potency of 8-[(phenylethyl)sulfanyl]caffeine. The results of the MAO inhibitory studies with the 8- [(phenylpropyl)sulfanyl]caffeine analogues showed that these compounds are also inhibitors of MAO-B with IC50 values of 0.061–0.500 μM. Those homologues substituted with chlorine on the para and meta positions of the phenyl ring were found to be exceptionally potent inhibitors with IC50 values of 0.061 μM and 0.062 μM, respectively. For the series of 8- (benzylsulfanyl)caffeines, meta substitution with chlorine (IC50 = 0.227 μM) and bromine (IC50 = 0.199 μM) was also found to enhance the MAO-B inhibition potency of 8- (benzylsulfanyl)caffeine (IC50 = 1.86 μM). The results document that the 8-sulfinylcaffeines are also inhibitors of MAO-B with IC50 values of 11.8–131 μM. The 8-sulfonylcaffeine was also found to be a MAO-B inhibitor. Compared to the 8-sulfanylcaffeines, these homologues are, however, weaker inhibitors. It may, therefore, be concluded that 8-sulfinylcaffeines and 8-sulfonylcaffeines are comparatively weak MAO-B inhibitors and less suited for the design of high potency MAO-B inhibitors. The results also document that the 8-[(phenylethyl)sulfanyl]caffeines are relatively weak MAO-A inhibitors with IC50 values of 5.66–141 μM, with one homologue exhibiting no inhibition under the experimental conditions. As evident from the selectivity indices (SI values), the 8- [(phenylethyl)sulfanyl]caffeines were all selective inhibitors of the MAO-B isoform. Two compounds exhibited SI values in excess of 1000. Since these compounds are also highly potent MAO-B inhibitors, they represent suitable leads for the design of potent and selective MAO-B inhibitors. The 8-sulfinylcaffeines and 8-sulfonylcaffeine were found to be weak MAO-A inhibitors with IC50 values of 166–250 μM. The SI values demonstrate that these compounds are MAO-B selective inhibitors, although to a lesser degree than the 8- [(phenylethyl)sulfanyl]caffeines. The 8-[(phenylpropyl)sulfanyl]caffeines are also MAO-A inhibitors with IC50 values of 0.708–6.48 μM. It is noteworthy that these homologues are the most potent MAO-A inhibitors among the compounds evaluated in this study. In fact, one of the 8-[(phenylpropyl)sulfanyl]caffeines, 8-{[3-(4-chlorophenyl)propyl]sulfanyl}caffeine (IC50 = 0.708 μM), is the only compound with an IC50 value for the inhibition of MAO-A in the submicromolar range. The 8-[(phenylpropyl)sulfanyl]caffeines display, in general, lower degrees of selectivity for MAO-B than the corresponding 8-[(phenylethyl)sulfanyl]caffeines. Reversibility studies: The reversibility of the interaction of a representative inhibitor, 8-{[2-(3- (trifluoromethyl)phenyl)ethyl]sulfanyl}caffeine, with MAO-B was investigated by evaluating the recovery of the enzymatic activity after dilution of the enzyme-inhibitor complex. For this purpose, MAO-B was preincubated with the test compound at concentrations of 10 × IC50 and 100 × IC50 for 30 min. The reactions were subsequently diluted 100-fold to 0.1 × IC50 and 1 × IC50, respectively. The results show that, after dilution to 0.1 × IC50 and 1 × IC50, the MAO-B catalytic activities are recovered to 35% and 22%, respectively, of the control value. For reversible enzyme inhibition, the enzyme activities are expected to recover to levels of approximately 90% and 50%, respectively, after 100-fold dilution of the preincubations containing inhibitor concentrations of 10 × IC50 and 100 × IC50. After preincubation of MAO-B with the irreversible inhibitor (R)-deprenyl (at 10 × IC50), and dilution of the resulting complex to 0.1 × IC50, MAO-B activity is not recovered (3.0% of control). These data indicate that the test compound does indeed react reversibly with MAO-B but because enzyme activities are not recovered to the expected 90% and 50% respectively, it may suggest that the test compound possess a quasi-reversible or tight-binding component. Hansch-type structure activity relationship studies: A limited Hansch-type QSAR study was performed for the inhibition of MAO by the 8-[(phenylethyl)sulfanyl]caffeines. For this purpose, five parameters were used to describe the physicochemical properties of the C-3 substituents on the phenyl rings of the inhibitors. The Van der Waals volume (Vw) and Taft steric parameter (Es) served as descriptors of the bulkiness of the substituents, while the lipophilicities were described by the Hansch constant (π). The electronic properties were described by the classical Hammett constant (σm) and the Swain-Lupton constant (F). A one-parameter fit with the Taft steric parameter versus the inhibition potency (logIC50) yielded the best correlation with a correlation coefficient (R2) of 0.912 and a statistical F value of 41.27 (Fmax = 35). The positive sign of the Es (+0.47) parameter coefficient indicated that the inhibition potencies of the 8- [(phenylethyl)sulfanyl]caffeines towards MAO-B may be enhanced by substitution with sterically large groups at C-3 of the phenyl rings of the inhibitors. / Thesis (MSc (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2013

Parkinson’s disease (PD) is an age-related neurodegenerative disorder. The degeneration of the neurons of the substantia nigra in the midbrain leads to the loss of dopamine from the striatum, which is responsible for the motor symptoms of PD. In the brain, the enzyme, monoamine oxidase B (MAOB), An analysis of the Lineweaver-Burk plots indicated that 7-(3-bromobenzyloxy)-3,4-dihydro-2(1H)- quinolinone inhibits MAO-B with a Ki value of 2.7 nM. An analysis of the structure-activity relationships for MAO-B inhibition shows that substitution on the C7 position of the 3,4-dihydro- 2(1H)-quinolinone moiety leads to significantly more potent inhibition compared to substitution on C6. In this regard, a benzyloxy substituent on C7 is more favourable than phenylethoxy and phenylpropoxy substitution on this position. In spite of this, C6-substituted 3,4-dihydro-2(1H)-quinolinone with potent MAO-B inhibitory activities were also identified. An analyses of selected properties of the 3,4-dihydro-2(1H)- quinolinones showed that the compounds are highly lipophilic with logP values in the range of 3.03- 4.55. LogP values between 1 and 3 are, however, in the ideal range for bioavailability. The compounds synthesised have logP values higher than 3, which may lead to lower bioavailability. Laboratory data further showed that none of the 3,4-dihydro-2(1H)-quinolinones are highly toxic to cultured cells at the concentrations, 1 μM and 10 μM, tested. For example, the most potent MAO-B inhibitor, 7-(3-bromobenzyloxy)-3,4-dihydro-2(1H)-quinolinone, reduced cell viability to 88.11% and 86.10% at concentrations of 1 μM and 10 μM, respectively. These concentrations are well above its IC50 value for the inhibition of MAO-B. At concentrations required for MAO-B inhibition, the more potent 3,4-dihydro-2(1H)-quinolinones are thus unlikely to be cytotoxic. It may thus be concluded that C7-substituted 3,4-dihydro-2(1H)-quinolinones are promising highly potent and selective MAO-B inhibitors, and thus leads for the therapy of Parkinson’s disease. represents a major catabolic pathway of dopamine. Inhibitors of MAO-B conserve the depleted supply of dopamine and are thus used in the therapy of PD. In the present study, a series of 3,4- dihydro-2(1H)-quinolinone derivatives were synthesized and evaluated as inhibitors of recombinant human MAO-A and MAO-B. These quinolinone derivatives are structurally related to a series of coumarin (1-benzopyran-2-one) derivatives, which has been reported to act as MAO-B inhibitors. C6- and C7-substituted 3,4-dihydro-2(1H)-quinolinone derivatives were synthesized by reacting 6- or 7- hydroxy-3,4-dihydro-2(1H)-quinolinone with an appropriately substituted alkyl bromide in the presence of base. To evaluate the MAO inhibitory properties (IC50 values) of the quinolinone derivatives the recombinant human MAO-A and MAO-B enzymes were used. The reversibility of inhibition of a representative 3,4-dihydro-2(1H)-quinolinone derivative was examined by measuring the recovery of enzyme activity after the dilution of the enzyme-inhibitor complexes, while the mode of MAO inhibition was determined by constructing Lineweaver-Burk plots. To determine the lipophilicity of the 3,4-dihydro-2(1H)-quinolinone derivatives, the logP values were measured. The toxicity of the 3,4-dihydro-2(1H)-quinolinone derivatives towards cultured cells (cytotoxicity) was also measured. The results document that the 3,4-dihydro-2(1H)-quinolinone derivatives are highly potent and selective MAO-B inhibitors with most homologues exhibiting IC50 values in the nanomolar range. The most potent MAO-B inhibitor, 7-(3-bromobenzyloxy)-3,4-dihydro-2(1H)-quinolinone, exhibits an IC50 value of 2.9 nM with a 2750-fold selectivity for MAO-B over the MAO-A isoform. As a MAO-B inhibitor, this compound is approximately equipotent to the most potent coumarin derivative (IC50 = 1.14 nM) reported in literature. Since MAO-B activity could be recovered after dilution of enzyme-inhibitor mixtures, it may be concluded that 7-(3-bromobenzyloxy)-3,4-dihydro-2(1H)- quinolinone is a reversible MAO-B inhibitor. The Lineweaver-Burk plots constructed for the inhibition of MAO-B by 7-(3-bromobenzyloxy)-3,4-dihydro-2(1H)-quinolinone were linear and intersected on the y-axis. These data indicated that this compound also is a competitive MAO-B inhibitor. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014

Parkinson’s disease (PD) is a neurodegenerative disorder, which is progressive in nature and usually associated with the elderly. It is the second most common age-related neurodegenerative disorder after Alzheimer’s disease (AD). PD occurs when there is a dramatic loss of dopamine (DA) in the striatum, a substructure of the basal ganglia, of the brain due to the degeneration of the nigrostriatal pathway that contains the dopaminergic neurons. Motor symptoms of PD include bradykinesia, muscular rigidity and resting tremors. Non-motor symptoms include speech and sleep problems, hallucinations and depression. Diverse treatment options are available to treat the symptoms of PD, including levodopa (L-Dopa), DA agonists and monoamine oxidase B (MAO-B) inhibitors. The MAOs are flavoproteins that are bound to the outer membrane of the mitochondria and catalyze the oxidative deamination of neurotransmitters such as serotonin (5-HT), noradrenaline (NA) and DA. Two isoforms occur, namely MAO-A and –B, which share a 70% sequence identity. MAO-A catalyzes the oxidation of 5-HT and MAO-B has a substrate specificity towards benzylamine and 2-phenylethylamine. DA, NA, adrenaline and tryptamine are oxidized by both forms. MAO-A plays an important role in depression while MAO-B plays an important role in PD. The two isoforms are not evenly distributed in the brain. Of particular relevance to PD is the observation that, in the basal ganglia, MAO-B is the predominant form and the oxidation of DA in this region is largely due to MAO-B activity. Also, with an increase in age, there is an up to fourfold increase in MAO-B activity in the brain. In the aged parkinsonian brain, MAO-B is therefore a major DA metabolizing enzyme and MAO-B inhibitors have an important role in the therapy of PD. MAO-B inhibitors may potentially reduce the metabolic destruction of DA and thereby provide relief from the symptoms of PD. MAO-B inhibitors may also exert a neuroprotective effect in PD. In the catalytic cycle of MAO-B, one mole each of an aldehyde, hydrogen peroxide and ammonia are formed for each mole of primary amine substrate oxidized. Ferrous iron, which is abundant in the basal ganglia, may react with the hydrogen peroxide to form hydroxyl radicals in the Fenton reaction. The hydroxyl radical damages virtually all types of biomolecules including proteins, DNA, lipids, carbohydrates and amino acids. The aldehyde, in turn, may react with amino groups of proteins, and thus lead to cell injury. Inhibitors of MAO-B may reduce the MAO-catalyzed formation of hydrogen peroxide and aldehydes in the basal ganglia, and thus act as neuroprotective agents. MAO-B inhibitors that are currently being used in the treatment of PD are selegiline and rasagiline. Both are irreversible inhibitors of MAO-B. While irreversible inhibitors of MAO have been used extensively as drugs, irreversible inhibition has a number of disadvantages. These include the loss of selectivity as a result of repeated drug administration and a slow and variable rate of enzyme recovery following termination of drug treatment. The turnover rate for the biosynthesis of MAO-B in the human brain may require as much as 40 days while with reversible inhibition, enzyme activity is recovered when the inhibitor is eliminated from the tissues. For these reasons the discovery of novel MAO-B inhibitors, which interact reversibly with the enzymes are of value in the therapy of PD. The goal of this study was to design novel and reversible inhibitors of MAO-B, which may find application in the therapy of PD. In the current study, caffeine was used as scaffold for the design of new MAO inhibitors. Caffeine is reported to be a weak inhibitor of MAO-B, with an IC50 value of 5084 μM. Substitution at C-8 of the caffeine moiety, however, yields compounds with potent MAO-B inhibitory properties. Of particular importance to this study is a recent report that a series of 8-sulfanylcaffeine analogues acts as selective inhibitors of human MAO-B. Among the compounds examined, 8-[(phenylethyl)sulfanyl]caffeine was found to be a particularly potent MAO-B inhibitor with an IC50 value of 0.223 μM. In an attempt to further enhance the MAO-B inhibition potency of 8-[(phenylethyl)sulfanyl]caffeine, and possibly to discover highly potent MAO-B inhibitors, a series of five 8-[(phenylethyl)sulfanyl]caffeine analogues was synthesized and evaluated as inhibitors of human MAO-A and –B. For the purpose of this study 8- [(phenylethyl)sulfanyl]caffeine homologues containing C-3 alkyl (CF3, CH3 and OCH3) and halogen (Cl and Br) substituents on the phenyl ring were considered. Furthermore, a series of two 8-sulfinylcaffeine analogues and one 8-sulfonylcaffeine were synthesized and their MAO inhibitory potencies were measured. The purpose with these compounds was to compare the MAO inhibitory properties of the 8-sulfinylcaffeine analogues and 8-sulfonylcaffeine with those of the 8-sulfanylcaffeine analogues. This study also investigates the MAO inhibition properties of three selected 8-[(phenylpropyl)sulfanyl]caffeine and two 8-(benzylsulfanyl)caffeine analogues. Chemistry: The target 8-sulfanylcaffeine analogues were synthesized according to the literature procedure. 8-Chlorocaffeine was reacted with an appropriate mercaptan in the presence of NaOH, to yield the target 8-sulfanylcaffeine analogues in yields of 6.4–50.7%. 8-Chlorocaffeine, in turn, was conveniently synthesized in high yield by reacting chlorine with caffeine in chloroform. In certain instances, the mercaptan starting materials were not commercially available and were thus synthesized according to the literature procedure by reacting an appropriate alkylbromide with thiourea. The resulting thiouronium salt was hydrolyzed in the presence of NaOH to yield the target mercaptan. The 8-sulfinylcaffeine analogues and 8- sulfonylcaffeine were synthesized by reacting the 8-sulfanylcaffeines with H2O2 in the presence of glacial acetic acid and acetic anhydride. The structures and the purities of the inhibitors were verified by NMR, MS and HPLC analyses. MAO inhibition studies: The MAO inhibitory properties of the test compounds were examined using the recombinant human enzymes. The mixed MAO-A/B substrate, kynuramine, was employed as substrate for both enzymes and the inhibition potencies were expressed as the IC50 values. The 8-[(phenylethyl)sulfanyl]caffeine analogues were found to be highly potent inhibitors of MAO-B. The IC50 values recorded for these homologues ranged from 0.017–0.125 μM, making them twofold to 13-fold more potent MAO-B inhibitors than the lead compound, 8- [(phenylethyl)sulfanyl]caffeine (IC50 = 0.223 μM). For comparison, the reversible MAO-B selective inhibitor, lazabemide, exhibits an IC50 value of 0.091 μM under the same conditions (unpublished data from our laboratory). Interestingly, both alkyl (CF3, CH3 and OCH3) and halogen (Cl and Br) substitution lead to highly potent MAO-B inhibition. It may therefore be concluded that substitution on C-3 is a general strategy to enhance the MAO-B inhibition potency of 8-[(phenylethyl)sulfanyl]caffeine. The results of the MAO inhibitory studies with the 8- [(phenylpropyl)sulfanyl]caffeine analogues showed that these compounds are also inhibitors of MAO-B with IC50 values of 0.061–0.500 μM. Those homologues substituted with chlorine on the para and meta positions of the phenyl ring were found to be exceptionally potent inhibitors with IC50 values of 0.061 μM and 0.062 μM, respectively. For the series of 8- (benzylsulfanyl)caffeines, meta substitution with chlorine (IC50 = 0.227 μM) and bromine (IC50 = 0.199 μM) was also found to enhance the MAO-B inhibition potency of 8- (benzylsulfanyl)caffeine (IC50 = 1.86 μM). The results document that the 8-sulfinylcaffeines are also inhibitors of MAO-B with IC50 values of 11.8–131 μM. The 8-sulfonylcaffeine was also found to be a MAO-B inhibitor. Compared to the 8-sulfanylcaffeines, these homologues are, however, weaker inhibitors. It may, therefore, be concluded that 8-sulfinylcaffeines and 8-sulfonylcaffeines are comparatively weak MAO-B inhibitors and less suited for the design of high potency MAO-B inhibitors. The results also document that the 8-[(phenylethyl)sulfanyl]caffeines are relatively weak MAO-A inhibitors with IC50 values of 5.66–141 μM, with one homologue exhibiting no inhibition under the experimental conditions. As evident from the selectivity indices (SI values), the 8- [(phenylethyl)sulfanyl]caffeines were all selective inhibitors of the MAO-B isoform. Two compounds exhibited SI values in excess of 1000. Since these compounds are also highly potent MAO-B inhibitors, they represent suitable leads for the design of potent and selective MAO-B inhibitors. The 8-sulfinylcaffeines and 8-sulfonylcaffeine were found to be weak MAO-A inhibitors with IC50 values of 166–250 μM. The SI values demonstrate that these compounds are MAO-B selective inhibitors, although to a lesser degree than the 8- [(phenylethyl)sulfanyl]caffeines. The 8-[(phenylpropyl)sulfanyl]caffeines are also MAO-A inhibitors with IC50 values of 0.708–6.48 μM. It is noteworthy that these homologues are the most potent MAO-A inhibitors among the compounds evaluated in this study. In fact, one of the 8-[(phenylpropyl)sulfanyl]caffeines, 8-{[3-(4-chlorophenyl)propyl]sulfanyl}caffeine (IC50 = 0.708 μM), is the only compound with an IC50 value for the inhibition of MAO-A in the submicromolar range. The 8-[(phenylpropyl)sulfanyl]caffeines display, in general, lower degrees of selectivity for MAO-B than the corresponding 8-[(phenylethyl)sulfanyl]caffeines. Reversibility studies: The reversibility of the interaction of a representative inhibitor, 8-{[2-(3- (trifluoromethyl)phenyl)ethyl]sulfanyl}caffeine, with MAO-B was investigated by evaluating the recovery of the enzymatic activity after dilution of the enzyme-inhibitor complex. For this purpose, MAO-B was preincubated with the test compound at concentrations of 10 × IC50 and 100 × IC50 for 30 min. The reactions were subsequently diluted 100-fold to 0.1 × IC50 and 1 × IC50, respectively. The results show that, after dilution to 0.1 × IC50 and 1 × IC50, the MAO-B catalytic activities are recovered to 35% and 22%, respectively, of the control value. For reversible enzyme inhibition, the enzyme activities are expected to recover to levels of approximately 90% and 50%, respectively, after 100-fold dilution of the preincubations containing inhibitor concentrations of 10 × IC50 and 100 × IC50. After preincubation of MAO-B with the irreversible inhibitor (R)-deprenyl (at 10 × IC50), and dilution of the resulting complex to 0.1 × IC50, MAO-B activity is not recovered (3.0% of control). These data indicate that the test compound does indeed react reversibly with MAO-B but because enzyme activities are not recovered to the expected 90% and 50% respectively, it may suggest that the test compound possess a quasi-reversible or tight-binding component. Hansch-type structure activity relationship studies: A limited Hansch-type QSAR study was performed for the inhibition of MAO by the 8-[(phenylethyl)sulfanyl]caffeines. For this purpose, five parameters were used to describe the physicochemical properties of the C-3 substituents on the phenyl rings of the inhibitors. The Van der Waals volume (Vw) and Taft steric parameter (Es) served as descriptors of the bulkiness of the substituents, while the lipophilicities were described by the Hansch constant (π). The electronic properties were described by the classical Hammett constant (σm) and the Swain-Lupton constant (F). A one-parameter fit with the Taft steric parameter versus the inhibition potency (logIC50) yielded the best correlation with a correlation coefficient (R2) of 0.912 and a statistical F value of 41.27 (Fmax = 35). The positive sign of the Es (+0.47) parameter coefficient indicated that the inhibition potencies of the 8- [(phenylethyl)sulfanyl]caffeines towards MAO-B may be enhanced by substitution with sterically large groups at C-3 of the phenyl rings of the inhibitors. / Thesis (MSc (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2013

Very little is known about why and how the Parkinson's disease (PD) neurodegenerative process begins and progresses. In the course of developments for treatment of PD, the discovery of the inhibition of monoamine oxidase (MAO B) was a conceptual breakthrough, and has now been firmly established. MAO B has also been implicated in the neurodegenerative processes resulting from exposure to xenobiotic amines. For example, MAO B catalyzes the first step of the bioactivation of the parkinsonian inducing pro-neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Additional insight into the mechanism of catalysis of MAO B and the mechanism of neurotoxicity by MPTP is therefore very valuable in the pursuit of the treatment of PD. / Thesis (Ph.D. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2008.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder, characterized pathologically by a marked loss of dopaminergic nigrostriatal neurons and clinically by disabling movement disorders. PD can be treated by inhibiting monoamine oxidase (MAO), specifically MAO–B, since this is a major enzyme involved in the catabolism of dopamine in the substantia nigra of the brain. Inhibition of MAO–B may conserve the dopamine supply in the brain and may therefore provide symptomatic relief for PD patients. Selegiline is an irreversible MAO–B inhibitor and is currently used for the treatment of PD. Irreversible inhibitors inactivate enzymes by forming stable covalent complexes. The process is not readily reversed either by removing the remainder of the free inhibitor or by increasing the substrate concentration. Even dilution or dialysis does not dissociate the enzyme inhibitor complex and restore enzyme activity. From a safety point of view it may therefore be more desirable to develop reversible inhibitors of MAO–B. In this study, caffeine was used as lead compound to design, synthesize and evaluate new reversible inhibitors of MAO–B. This study is based on the finding that C–8 substituted caffeine analogues are potent MAO inhibitors. For example, (E)–8–(3–chlorostyryl)caffeine (CSC) is an exceptionally potent competitive inhibitor of MAO–B with an enzyme–inhibitor dissociation constant (Ki value) of 128 nM. In this study caffeine was similarly conjugated at C–8 to various side–chains. The effect that these chosen side–chains had on the MAO–B inhibition activity of C–8 substituted caffeine analogues will then be evaluated. The caffeine analogues were also evaluated as human MAO–A inhibitors. For the purpose of this study, saturated C–8 side chains were selected with the goal of discovering new C–8 side chains that enhance the MAO–A and ?B inhibition potency of caffeine. As mentioned above, the styryl side chain is one example of a side chain that enhances the MAO–B inhibition potency of caffeine. Should a side chain with promising MAO inhibition activity be identified in this study, the inhibition potency will be further optimized in a future study by the addition of a variety of substituents to the C–8 side chain ring. For example, halogen substitution of (E)–8– styrylcaffeine enhances the MAO–B inhibition potency by up to 10 fold. The saturated side chains selected for the present study included the phenylethyl (1), phenylpropyl (2), phenylbutyl (3) and phenylpentyl (4) functional groups. Also included are the cyclohexylethyl (8), 3–oxo–3– phenylpropyl (5), 4–oxo–4–phenylbutyl (6) moieties. A test compound containing an unsaturated linker between C–8 of caffeine and the side chain ring, the phenylpropenyl analogue 7, was also included. This study is therefore an exploratory study to discover new C–8 moieties that are favorable for MAO– inhibition. All the target compounds were synthesized by reacting 1,3–dimethyl–5,6–diaminouracil with an appropriate carboxylic acid in the presence of a carbodiimide dehydrating agent. Following ring closure and methylation at C–7, the target inhibitors were obtained. Inhibition potencies were determined using recombinant human MAO–A and MAO–B as enzyme sources. The inhibitor potencies were expressed as IC50 values. The most potent MAO–B inhibitor was 8–(5– phenylpentyl)caffeine (4) with an IC50 value of 0.656 ?M. In contrast, all the other test inhibitors were moderately potent MAO–B inhibitors. In fact the next best MAO–B inhibitor, 8–(4– phenylbutyl)caffeine (3) was approximately 5 fold less potent than 4 with an IC50 value of 3.25 ?M. Since the 5–phenylpentyl moiety is the longest side chain evaluated in this study, this finding demonstrates that longer C–8 side chains are more favorable for MAO–B inhibition. Interestingly, compound 5 containing a cyclohexylethyl side chain (IC50 = 6.59 ?M) was approximately 4 fold more potent than the analogue containing the phenylethyl linker (1) (IC50 = 26.0 ?M). This suggests that a cyclohexyl ring in the C–8 side chain of caffeine may be more optimal for MAO–B inhibition and should be considered in future studies. The caffeine analogues containing the oxophenylalkyl side chains (5 and 6) were weak MAO–B inhibitors with IC50 values of 187 ?M and 46.9 ?M, respectively. This suggests that the presence of a carbonyl group in the C–8 side chain is not favorable for the MAO–B inhibition potency of caffeine. The unsaturated phenylpropenyl analogue 7 was also found to be a relatively weak MAO–B inhibitor with an IC50 value of 33.1 ?M. In contrast to the results obtained with MAO–B, the test caffeine analogues were all weak MAOA inhibitors. With the exception of compound 5, all of the analogues evaluated were selective inhibitors of MAO–B. The most potent MAO–B inhibitor, 8–(5–phenylpentyl)caffeine (4) was the most selective inhibitor, 48 fold more potent towards MAO–B than MAO–A. This study also shows that two selected analogues (5 and 3) bind reversibly to MAO–A and ?B, respectively, and that the mode of MAO–A and –B inhibition is competitive for these representative compounds. / Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2011.

Very little is known about why and how the Parkinson's disease (PD) neurodegenerative process begins and progresses. In the course of developments for treatment of PD, the discovery of the inhibition of monoamine oxidase (MAO B) was a conceptual breakthrough, and has now been firmly established. MAO B has also been implicated in the neurodegenerative processes resulting from exposure to xenobiotic amines. For example, MAO B catalyzes the first step of the bioactivation of the parkinsonian inducing pro-neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Additional insight into the mechanism of catalysis of MAO B and the mechanism of neurotoxicity by MPTP is therefore very valuable in the pursuit of the treatment of PD. / Thesis (Ph.D. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2008.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder, characterized pathologically by a marked loss of dopaminergic nigrostriatal neurons and clinically by disabling movement disorders. PD can be treated by inhibiting monoamine oxidase (MAO), specifically MAO–B, since this is a major enzyme involved in the catabolism of dopamine in the substantia nigra of the brain. Inhibition of MAO–B may conserve the dopamine supply in the brain and may therefore provide symptomatic relief for PD patients. Selegiline is an irreversible MAO–B inhibitor and is currently used for the treatment of PD. Irreversible inhibitors inactivate enzymes by forming stable covalent complexes. The process is not readily reversed either by removing the remainder of the free inhibitor or by increasing the substrate concentration. Even dilution or dialysis does not dissociate the enzyme inhibitor complex and restore enzyme activity. From a safety point of view it may therefore be more desirable to develop reversible inhibitors of MAO–B. In this study, caffeine was used as lead compound to design, synthesize and evaluate new reversible inhibitors of MAO–B. This study is based on the finding that C–8 substituted caffeine analogues are potent MAO inhibitors. For example, (E)–8–(3–chlorostyryl)caffeine (CSC) is an exceptionally potent competitive inhibitor of MAO–B with an enzyme–inhibitor dissociation constant (Ki value) of 128 nM. In this study caffeine was similarly conjugated at C–8 to various side–chains. The effect that these chosen side–chains had on the MAO–B inhibition activity of C–8 substituted caffeine analogues will then be evaluated. The caffeine analogues were also evaluated as human MAO–A inhibitors. For the purpose of this study, saturated C–8 side chains were selected with the goal of discovering new C–8 side chains that enhance the MAO–A and ?B inhibition potency of caffeine. As mentioned above, the styryl side chain is one example of a side chain that enhances the MAO–B inhibition potency of caffeine. Should a side chain with promising MAO inhibition activity be identified in this study, the inhibition potency will be further optimized in a future study by the addition of a variety of substituents to the C–8 side chain ring. For example, halogen substitution of (E)–8– styrylcaffeine enhances the MAO–B inhibition potency by up to 10 fold. The saturated side chains selected for the present study included the phenylethyl (1), phenylpropyl (2), phenylbutyl (3) and phenylpentyl (4) functional groups. Also included are the cyclohexylethyl (8), 3–oxo–3– phenylpropyl (5), 4–oxo–4–phenylbutyl (6) moieties. A test compound containing an unsaturated linker between C–8 of caffeine and the side chain ring, the phenylpropenyl analogue 7, was also included. This study is therefore an exploratory study to discover new C–8 moieties that are favorable for MAO– inhibition. All the target compounds were synthesized by reacting 1,3–dimethyl–5,6–diaminouracil with an appropriate carboxylic acid in the presence of a carbodiimide dehydrating agent. Following ring closure and methylation at C–7, the target inhibitors were obtained. Inhibition potencies were determined using recombinant human MAO–A and MAO–B as enzyme sources. The inhibitor potencies were expressed as IC50 values. The most potent MAO–B inhibitor was 8–(5– phenylpentyl)caffeine (4) with an IC50 value of 0.656 ?M. In contrast, all the other test inhibitors were moderately potent MAO–B inhibitors. In fact the next best MAO–B inhibitor, 8–(4– phenylbutyl)caffeine (3) was approximately 5 fold less potent than 4 with an IC50 value of 3.25 ?M. Since the 5–phenylpentyl moiety is the longest side chain evaluated in this study, this finding demonstrates that longer C–8 side chains are more favorable for MAO–B inhibition. Interestingly, compound 5 containing a cyclohexylethyl side chain (IC50 = 6.59 ?M) was approximately 4 fold more potent than the analogue containing the phenylethyl linker (1) (IC50 = 26.0 ?M). This suggests that a cyclohexyl ring in the C–8 side chain of caffeine may be more optimal for MAO–B inhibition and should be considered in future studies. The caffeine analogues containing the oxophenylalkyl side chains (5 and 6) were weak MAO–B inhibitors with IC50 values of 187 ?M and 46.9 ?M, respectively. This suggests that the presence of a carbonyl group in the C–8 side chain is not favorable for the MAO–B inhibition potency of caffeine. The unsaturated phenylpropenyl analogue 7 was also found to be a relatively weak MAO–B inhibitor with an IC50 value of 33.1 ?M. In contrast to the results obtained with MAO–B, the test caffeine analogues were all weak MAOA inhibitors. With the exception of compound 5, all of the analogues evaluated were selective inhibitors of MAO–B. The most potent MAO–B inhibitor, 8–(5–phenylpentyl)caffeine (4) was the most selective inhibitor, 48 fold more potent towards MAO–B than MAO–A. This study also shows that two selected analogues (5 and 3) bind reversibly to MAO–A and ?B, respectively, and that the mode of MAO–A and –B inhibition is competitive for these representative compounds. / Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2011.

Fundação de Amparo a Pesquisa no Estado do Rio Grande do Sul / Depression is a complex, chronic and disabling psychiatric disease that carries a high social cost. Among the various classes of antidepressants are the monoamine oxidase A (MAO-A) inhibitors that reduce monoamine metabolism. An important site of MAO-A regulation is the imidazoline-2 binding site (I2). In fact, it was recently shown that 2-imidazoline derivatives, such as 2-(2-benzofuranyl)-2-imidazoline (2- BFI), showed good potency and selectivity in inhibiting in vitro the activity of MAO-A, but the antidepressant potential of this compound and its mechanism of action have not been well defined. Therefore, in this study we investigated the antidepressant-like effect of 2-BFI in mice. For this purpose, we evaluated the effects of 2-BFI in two predictive tests of antidepressant-like activity in animals, the tail suspension test (TST) and forced swimming test (FST). The TSC was utilized after the use of specific antagonists of different receptors involved in depression. 2-BFI (100 and 300 μmol/kg, s.c.) significantly reduced the immobility time on the tail suspension test (TST) without changing locomotion in the open field test. The reduced the immobility time of 2-BFI (100 μmol/kg, s.c.) was confirmed with the forced swimming test (FST). The antidepressant-like effect of 2-BFI (100 μmol/kg, s.c.) in the TST was prevented by pretreatment with idazoxan (0.4 μmol/kg, i.p., a I2 site antagonist), methysergide (4 μmol/kg, i.p., a non-selective serotonergic receptor antagonist) and haloperidol (0.1 μmol/kg, i.p., a non-selective dopaminergic receptor antagonist). The anxiolytic effect of 2-BFI was also evaluated, using the elevated plus-maze test. 2-BFI (300 μmol/kg, s.c.) was able to significantly increase the % of number of entries and the % of time spent in the open arms, indicating that it possesses an anxiolytic effect at high doses. In conclusion, these results suggest that the antidepressant-like effect of 2- BFI might involve serotonergic, dopaminergic and imidazoline systems, and then the imidazoline site could represent a new pharmacological target for the treatment of depression. / A depressão é uma doença psiquiátrica complexa, crônica e incapacitante, que acarreta um alto custo social. Dentre as diversas classes de antidepressivos encontram-se os inibidores da monoamina oxidase-A (MAO-A), que reduzem o metabolismo das monoaminas. Um sítio importante para a regulação da MAO-A é o sítio imidazolínico I2. Recentemente, foi demonstrado que derivados 2- imidazolínicos, como o 2-BFI, mostram boa potência e seletividade em inibir a atividade in vitro da MAO em cérebro de ratos, porém o potencial antidepressivo deste composto e seu mecanismo de ação não foram bem definidos. Com base nisso, o objetivo desse trabalho consiste em investigar o efeito tipo-antidepressivo do 2-BFI em camundongos. Para este propósito, foram avaliados os efeitos do 2-BFI em dois testes preditivos de atividade antidepressiva em animais, o teste de suspensão da cauda (TSC) e o teste do nado forçado (TNF). O TSC também foi empregado após o uso de antagonistas específicos de diferentes receptores envolvidos na depressão. O 2-BFI (100 e 300 μmol/kg, s.c.) reduziu significativamente o tempo de imobilidade no TSC, sem alterar a atividade locomotora no teste de campo aberto. A redução do tempo de imobilidade de 2-BFI (100 μmol/kg, s.c.) foi confirmada com o TNF. O efeito tipo-antidepressivo do 2-BFI (100 μmol/kg, s.c.) no TSC foi prevenido pelo pré-tratamento com idazoxan (0,4 μmol/kg, i.p., um antagonista do sítio I2), metisergida (4 μmol/kg, i.p., um antagonista não-seletivo dos receptores serotoninérgicos) e haloperidol (0,1 μmol/kg, i.p., um antagonista não-seletivo dos receptores dopaminérgicos). O efeito ansiolítico do 2- BFI também foi avaliado, utilizando o teste de labirinto em cruz elevado. O 2-BFI (300 μmol/kg, s.c.) aumentou significativamente a % do número de entradas e a % do tempo gasto nos braços abertos, indicando que ele possui um efeito ansiolítico em altas doses. Em conclusão, estes resultados sugerem que o efeito tipoantidepressivo de 2-BFI pode estar envolvido com os sistemas serotoninérgico, dopaminérgico e imidazolínico, e assim o sítio imidazolínico poderia representar um novo alvo farmacológico para o tratamento da depressão.