Background and rationale: Parkinson’s disease (PD) is a progressive, degenerative disorder of the central nervous system and is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. The loss of functional dopamine in the striatum is thought to be responsible for the typical symptoms of PD. Cardinal features of PD include bradykinesia, muscular rigidity, resting tremor and impairment of postural balance. This study focuses on the inhibition of monoamine oxidase B (MAO-B) and antagonism of A2A receptors as therapeutic strategies for PD. Monoamine oxidase (MAO) is a flavin adenine dinucleotide (FAD)-containing mitochondrial bound isoenzyme which consists of two isoforms namely MAO-A and MAO-B. The primary function of MAO is to catalyze the oxidative deamination of dietary amines, monoamine neurotransmitters and hormones. MAO-A is responsible for the oxidative deamination of serotonin (5-HT) and norepinephrine (NE), while MAO-B is responsible for the oxidative deamination of dopamine (DA). The formation of DA takes place in the presynaptic neuron where it is stored in vesicles and released into the presynaptic cleft. The released DA then either binds to D1 and D2 receptors which results in an effector response. The excess DA in the presynaptic cleft is metabolized by MAO-B which may result in the formation of free radicals and a decrease in DA concentrations. Under normal physiological conditions free radicals are removed from the body via normal physiological processes, but in PD these normal physiological processes are thought to be unable to remove the radicals and this may lead to oxidative stress. Oxidative stress is believed to be one of the leading causes of neurodegeneration in PD. The rationale for the use of MAO-B inhibitors in PD would be to increase the natural DA levels in the brain and also diminish the likelihood of free radicals to be formed. Adenosine is an endogenous purine nucleoside and yields a variety of physiological effects. Four adenosine receptor subtypes have been characterized: A1, A2A, A2B and A3. They are all part of the G-protein-coupled receptor family and have seven transmembrane domains. The A2A receptor is highly concentrated in the striatum. There are two important pathways in the basal ganglia (BG) through which striatal information reaches the globus pallidus, namely the direct pathway containing A1 and D1 receptors and the indirect pathway containing A2A and D2 receptors. The direct pathway facilitates willed movement and the indirect pathway inhibits willed movement. A balance of the two pathways is necessary for normal movement. In PD, there is a decrease in DA in the striatum, thus leading to unopposed A2A receptor signaling and ultimately resulting in overactivity of the indirect pathway. Overactivity of the indirect pathway results in the locomotor symptoms associated with PD. Treatment with an A2A antagonist will block the A2A receptor, resulting in the restoration of balance between the indirect and direct pathways, thus leading to a decrease in locomotor symptoms. Aim: In this study, caffeine served as a lead compound for the design of dual-targeted drugs that are selective, reversible MAO-B inhibitors as well as A2A antagonists. Caffeine is a very weak MAO-B inhibitor and a moderately potent A2A antagonist. Substitution on the C8 position of caffeine yields compounds with good MAO-B inhibition activities and A2A receptor affinities. An example of this behaviour is found with (E)-8-(3-chlorostyryl)caffeine (CSC), which is not only a potent A2A antagonist but also a potent MAO-B inhibitor. The goal of this study was to identify and synthesize dual-targeted xanthine compounds. Recently Swanepoel and co-workers (2012) found that 8-phenoxymethyl substituted caffeines are potent reversible inhibitors of MAO-B. Therefore, this study focused on expanding the 8-(phenoxymethyl)caffeine series and evaluating the resulting compounds as both MAO-A and -B inhibitors as well as A2A antagonists. Synthesis: Two series were synthesized namely the 8-(phenoxymethyl)caffeines and 1,3-diethyl-7-methyl-8-(phenoxymethyl)xanthines. The analogues were synthesized according to the literature procedure. 1,3-Dimethyl-5,6-diaminouracil or 1,3-diethyl-5,6-diaminouracil were used as starting materials and were acylated with a suitable substituted phenoxyacetic acid in the presence of N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDAC) as an activating reagent. The intermediary amide was treated with sodium hydroxide, which resulted in ring closure to yield the corresponding 1,3-dimethyl-8-phenoxymethyl-7Hxanthinyl or 1,3-diethyl-8-phenoxymethyl-7H-xanthinyl analogues. These xanthines were 7-N-methylated in the presence of an excess of potassium carbonate and iodomethane to yield the target compounds. In vitro evaluation: A radioligand binding assay was performed to determine the affinities of the synthesized compounds for the A2A receptor. The MAO-B inhibition studies were carried out via a fluorometric assay where the MAO-catalyzed formation of H2O2 was measured. Results: Both series showed good to moderate MAO-B inhibition activities, while none of the compounds had activity towards MAO-A. Results were comparable to that of a known MAOB inhibitor lazabemide. For example, lazabemide (IC50 = 0.091 μM) was twice as potent as the most potent compound identified in this study, 8-(3-chlorophenoxymethyl)caffeine (compound 3; IC50 = 0.189 μM). Two additional compounds, 8-(4-iodophenoxymethyl)caffeine and 8-(3,4-dimethylphenoxymethyl) caffeine, also exhibited submicromolar IC50 values for the inhibition of MAO-B. The structure-activity relationships (SARs) indicated that 1,3-diethyl substitution resulted in decreased inhibition potency towards MAO-B and that 1,3-dimethyl substitution was a more suitable substitution pattern, leading to better inhibition potencies towards MAO-B. The compounds were also evaluated for A2A binding affinity, and relatively weak affinities were recorded with the most potent compound, 1,3-diethyl-7-methyl-8-[4-chlorophenoxymethyl]xanthine (compound 16), exhibiting a Ki value of 0.923 μM. Compared to KW-6002 (Ki = 7.94 nM), a potent reference A2A antagonist, compound 16 was 35-fold less potent. Comparing compound 16 to CSC [Ki(A2A) = 22.6 nM; IC50(MAO-B) = 0.146 nM], it was found that compound 16 is 31-fold less potent as an A2A antagonist and 21-fold less potent as a MAO-B inhibitor. Loss of MAO-B inhibition potency may be attributed to 1,3-diethyl substitution which correlates with similar conclusions reached in earlier studies. In addition, the replacement of the styryl functional group (as found with CSC and KW-6002) with the phenoxymethyl functional group (as found with the present series) may explain the general reduction in affinity for the A2A receptor. This suggests that the styryl side chain is more appropriate for A2A antagonism than the phenoxymethyl functional group. Conclusion: In this study two series of xanthine derivatives were successfully synthesized, namely the 8-(phenoxymethyl)caffeines and 1,3-diethyl-7-methyl-8-(phenoxymethyl)xanthines (11 compounds in total). Three of the newly synthesized compounds were found to act as potent inhibitors of MAO-B, with IC50 values in the submicromolar range. None of the compounds were however noteworthy MAO-A inhibitors. The most potent A2A antagonist among the examined compounds, compound 16, proved to be moderately potent compared to the reference antagonists, CSC and KW-6002. It may be concluded that the styryl functional group (as found with CSC and KW-6002) is more optimal than the phenoxymethyl functional group (as found with the present series) for A2A antagonism. 1,3-Diethyl substitution of the xanthine ring was found to be less optimal for MAO-B inhibition compared to 1,3-dimethyl substitution. These results together with known SARs provide valuable insight into the design of 8-(phenoxymethyl)caffeines as selective and potent MAO-B inhibitors. Such drugs may find application in the therapy of PD. / Thesis (MSc (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2013.

Background and rationale: Parkinson’s disease (PD) is a progressive, degenerative disorder of the central nervous system and is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. The loss of functional dopamine in the striatum is thought to be responsible for the typical symptoms of PD. Cardinal features of PD include bradykinesia, muscular rigidity, resting tremor and impairment of postural balance. This study focuses on the inhibition of monoamine oxidase B (MAO-B) and antagonism of A2A receptors as therapeutic strategies for PD. Monoamine oxidase (MAO) is a flavin adenine dinucleotide (FAD)-containing mitochondrial bound isoenzyme which consists of two isoforms namely MAO-A and MAO-B. The primary function of MAO is to catalyze the oxidative deamination of dietary amines, monoamine neurotransmitters and hormones. MAO-A is responsible for the oxidative deamination of serotonin (5-HT) and norepinephrine (NE), while MAO-B is responsible for the oxidative deamination of dopamine (DA). The formation of DA takes place in the presynaptic neuron where it is stored in vesicles and released into the presynaptic cleft. The released DA then either binds to D1 and D2 receptors which results in an effector response. The excess DA in the presynaptic cleft is metabolized by MAO-B which may result in the formation of free radicals and a decrease in DA concentrations. Under normal physiological conditions free radicals are removed from the body via normal physiological processes, but in PD these normal physiological processes are thought to be unable to remove the radicals and this may lead to oxidative stress. Oxidative stress is believed to be one of the leading causes of neurodegeneration in PD. The rationale for the use of MAO-B inhibitors in PD would be to increase the natural DA levels in the brain and also diminish the likelihood of free radicals to be formed. Adenosine is an endogenous purine nucleoside and yields a variety of physiological effects. Four adenosine receptor subtypes have been characterized: A1, A2A, A2B and A3. They are all part of the G-protein-coupled receptor family and have seven transmembrane domains. The A2A receptor is highly concentrated in the striatum. There are two important pathways in the basal ganglia (BG) through which striatal information reaches the globus pallidus, namely the direct pathway containing A1 and D1 receptors and the indirect pathway containing A2A and D2 receptors. The direct pathway facilitates willed movement and the indirect pathway inhibits willed movement. A balance of the two pathways is necessary for normal movement. In PD, there is a decrease in DA in the striatum, thus leading to unopposed A2A receptor signaling and ultimately resulting in overactivity of the indirect pathway. Overactivity of the indirect pathway results in the locomotor symptoms associated with PD. Treatment with an A2A antagonist will block the A2A receptor, resulting in the restoration of balance between the indirect and direct pathways, thus leading to a decrease in locomotor symptoms. Aim: In this study, caffeine served as a lead compound for the design of dual-targeted drugs that are selective, reversible MAO-B inhibitors as well as A2A antagonists. Caffeine is a very weak MAO-B inhibitor and a moderately potent A2A antagonist. Substitution on the C8 position of caffeine yields compounds with good MAO-B inhibition activities and A2A receptor affinities. An example of this behaviour is found with (E)-8-(3-chlorostyryl)caffeine (CSC), which is not only a potent A2A antagonist but also a potent MAO-B inhibitor. The goal of this study was to identify and synthesize dual-targeted xanthine compounds. Recently Swanepoel and co-workers (2012) found that 8-phenoxymethyl substituted caffeines are potent reversible inhibitors of MAO-B. Therefore, this study focused on expanding the 8-(phenoxymethyl)caffeine series and evaluating the resulting compounds as both MAO-A and -B inhibitors as well as A2A antagonists. Synthesis: Two series were synthesized namely the 8-(phenoxymethyl)caffeines and 1,3-diethyl-7-methyl-8-(phenoxymethyl)xanthines. The analogues were synthesized according to the literature procedure. 1,3-Dimethyl-5,6-diaminouracil or 1,3-diethyl-5,6-diaminouracil were used as starting materials and were acylated with a suitable substituted phenoxyacetic acid in the presence of N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDAC) as an activating reagent. The intermediary amide was treated with sodium hydroxide, which resulted in ring closure to yield the corresponding 1,3-dimethyl-8-phenoxymethyl-7Hxanthinyl or 1,3-diethyl-8-phenoxymethyl-7H-xanthinyl analogues. These xanthines were 7-N-methylated in the presence of an excess of potassium carbonate and iodomethane to yield the target compounds. In vitro evaluation: A radioligand binding assay was performed to determine the affinities of the synthesized compounds for the A2A receptor. The MAO-B inhibition studies were carried out via a fluorometric assay where the MAO-catalyzed formation of H2O2 was measured. Results: Both series showed good to moderate MAO-B inhibition activities, while none of the compounds had activity towards MAO-A. Results were comparable to that of a known MAOB inhibitor lazabemide. For example, lazabemide (IC50 = 0.091 μM) was twice as potent as the most potent compound identified in this study, 8-(3-chlorophenoxymethyl)caffeine (compound 3; IC50 = 0.189 μM). Two additional compounds, 8-(4-iodophenoxymethyl)caffeine and 8-(3,4-dimethylphenoxymethyl) caffeine, also exhibited submicromolar IC50 values for the inhibition of MAO-B. The structure-activity relationships (SARs) indicated that 1,3-diethyl substitution resulted in decreased inhibition potency towards MAO-B and that 1,3-dimethyl substitution was a more suitable substitution pattern, leading to better inhibition potencies towards MAO-B. The compounds were also evaluated for A2A binding affinity, and relatively weak affinities were recorded with the most potent compound, 1,3-diethyl-7-methyl-8-[4-chlorophenoxymethyl]xanthine (compound 16), exhibiting a Ki value of 0.923 μM. Compared to KW-6002 (Ki = 7.94 nM), a potent reference A2A antagonist, compound 16 was 35-fold less potent. Comparing compound 16 to CSC [Ki(A2A) = 22.6 nM; IC50(MAO-B) = 0.146 nM], it was found that compound 16 is 31-fold less potent as an A2A antagonist and 21-fold less potent as a MAO-B inhibitor. Loss of MAO-B inhibition potency may be attributed to 1,3-diethyl substitution which correlates with similar conclusions reached in earlier studies. In addition, the replacement of the styryl functional group (as found with CSC and KW-6002) with the phenoxymethyl functional group (as found with the present series) may explain the general reduction in affinity for the A2A receptor. This suggests that the styryl side chain is more appropriate for A2A antagonism than the phenoxymethyl functional group. Conclusion: In this study two series of xanthine derivatives were successfully synthesized, namely the 8-(phenoxymethyl)caffeines and 1,3-diethyl-7-methyl-8-(phenoxymethyl)xanthines (11 compounds in total). Three of the newly synthesized compounds were found to act as potent inhibitors of MAO-B, with IC50 values in the submicromolar range. None of the compounds were however noteworthy MAO-A inhibitors. The most potent A2A antagonist among the examined compounds, compound 16, proved to be moderately potent compared to the reference antagonists, CSC and KW-6002. It may be concluded that the styryl functional group (as found with CSC and KW-6002) is more optimal than the phenoxymethyl functional group (as found with the present series) for A2A antagonism. 1,3-Diethyl substitution of the xanthine ring was found to be less optimal for MAO-B inhibition compared to 1,3-dimethyl substitution. These results together with known SARs provide valuable insight into the design of 8-(phenoxymethyl)caffeines as selective and potent MAO-B inhibitors. Such drugs may find application in the therapy of PD. / Thesis (MSc (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2013.

Purpose: Monoamine oxidase (MAO) plays a key role in the treatment of Parkinson‟s disease (PD), since it is the major enzyme responsible for the catabolism of dopamine in the substantia nigra of the brain. Inhibition of MAO-B may conserve dopamine in the brain and provide symptomatic relief. The MAO-B inhibitors that are currently used for the treatment of PD, are associated with a variety of adverse effects (psychotoxic and cardiovascular effects) along with additional disadvantages such as irreversible inhibition of the enzyme. Irreversible inhibition may be considered a disadvantage, since following treatment with irreversible inhibitors, the rate by which the enzyme activity is recovered may be variable and may require several weeks. In contrast, following the administration of reversible inhibitors, enzyme activity is recovered when the inhibitor is cleared from the tissues. There exists therefore, a need to develop new reversible inhibitors of MAO-B which are considered to be safer than irreversible MAO-B inhibitors. Rationale: Recently discovered reversible MAO-B inhibitors include safinamide and (E)-8-(3-chlorostyryl)caffeine (CSC). Safinamide has a benzyloxy side chain, which is thought to be important for inhibition of MAO-B. CSC, on the other hand, consists of a caffeine moiety with a styryl substituent at C-8, which is also a critical feature for its inhibitory activity. In a previous study, the caffeine ring and the benzyloxy side chain were combined to produce a series of 8-benzyloxycaffeine analogues which proved to be potent new MAO-B inhibitors. In this study, caffeine was substituted with the phenoxymethyl functional group at C-8, instead of the benzyloxy moiety. The aim of this study was therefore to compare the MAO-B inhibition potencies of selected 8-(phenoxymethyl)caffeine analogues with the previously studied 8-benzyloxycaffeine analogues. In the current study, 8-(phenoxymethyl)caffeine (1) and nine 8-(phenoxymethyl)caffeine analogues (2-10) were synthesized and evaluated as inhibitors of recombinant human MAOA and –B. These analogues only differed in substitution on C3 and C4 of the phenoxymethyl phenyl ring. The substituents that were selected were halogens (Cl, F, and Br), the methyl group, the methoxy group and the trifluoromethyl group. These substituents are similar to those selected in a previous study where 8-benzyloxycaffeine analogues were evaluated as MAO inhibitors. This study therefore explores the effect that a variety of substituents on C3 and C4 of the phenoxymethyl phenyl ring will have on the MAO-A and –B inhibition potencies of 8-(phenoxymethyl)caffeine. Based on the results, additional 8-(phenoxymethyl)caffeine analogues with improved MAO-A and –B inhibition potencies will be proposed for investigation in future studies. Methods: The target, 8-(phenoxymethyl)caffeine, analogues were synthesized by reacting 1,3- dimethyl-5,6-diaminouracil with the appropriately substituted phenoxyacetic acid in the presence of a carbodiimide coupling agent. Ring closure was catalyzed in basic conditions and methylation of the resulting theophyline intermediates at C-7 was carried out with iodomethane. The structures and purities of all the target compounds were verified by NMR, MS and HPLC analysis. All of the 8-(phenoxymethyl)caffeine analogues were subsequently evaluated as MAO-A and –B inhibitors using the recombinant human enzymes. The inhibition potencies of the analogues were expressed as the IC50 values (concentration of the inhibitor that produces 50% inhibition). In addition, the time-dependency of inhibition of both MAO-A and –B was evaluated for two inhibitors in order to determine if these inhibitors interact reversibly or irreversibly with the MAO isozymes. A Hansch-type quantitative structure-activity relationship (QSAR) study was carried out in order to quantify the effect that different substituents on the phenyl ring of the 8-(phenoxymethyl)caffeine analogues have on MAO-B inhibition activity. Results: The results showed that among the test compounds, several analogues potently inhibited human MAO-B. The most potent inhibitor was 8-(3-bromophenoxymethyl)caffeine with an IC50 value of 0.148 μM toward human MAO-B. There were also inhibitors which displayed inhibition activities towards human MAO-A with IC50 values ranging from 4.59 μM to 34.0 μM. Compared to the 8-benzyloxycaffeine analogues, that were in general non-selective inhibitors, the 8-(phenoxymethyl)caffeine analogues, evaluated here, were selective for MAO-B. For example, 8-(3-bromophenoxymethyl)caffeine was found to be 141 fold more selective as an inhibitor of MAO-B than of MAO-A. Also, compared to the 8-benzyloxycaffeine analogues, the 8-(phenoxymethyl)caffeine analogues were slightly less potent MAO-B inhibitors. For example, 8-benzyloxycaffeine is reported to have an IC50 value of 1.77 μM for the inhibition of human MAO-B while 8-(phenoxymethyl)caffeine was found to have an IC50 value of 5.78 μM for the inhibition of human MAO-B. This study also shows that two selected analogues bind reversibly to MAO-A and –B, respectively, and that the mode of MAO-B inhibition is competitive for one representative compound. Qualitative inspection of the results revealed interesting structure-activity relationships. For the 8-(phenoxymethyl)caffeine analogues, bearing both the C3 and C4 substituents on the phenyl ring, the MAO-B activity significantly increases with halogen substitution. Furthermore, increased MAO-B inhibition was observed with increased electronegativity of the halogen substituent. To quantify these apparent relationships, a Hansch-type QSAR study was carried out. The results showed that the logarithm of the IC50 values (logIC50) correlated with Hansch lipophilicity (π) and the Swain-Lupton electronic (F) constants of the substituents at C-3 of the phenoxymethyl ring. The correlation exhibited an R2 value of 0.87 and a statistical F value of 13.6. From these results it may be concluded that electron-withdrawing substituents at C3 with a high degree of lipophilicity enhance MAO-B inhibition potency. These results are similar to those previously obtained for the series of 8-benzyloxycaffeine analogues. For this series, the MAO-B inhibition potencies correlated with the Hansch lipophilicity (π) and Hammett electronic (σ) constants of the substituents at C-3 of the benzyloxy ring. Similarly to the 8-(phenoxymethyl)caffeine analogues, electron-withdrawing substituents with a high degree of lipophilicity also enhance the MAO-B inhibition potencies of 8-benzyloxycaffeine analogues. / Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2011

Purpose Monoamine oxidase (MAO) consists of two isoforms, namely MAO-A and MAO-B. Both these isoforms are involved in the oxidation of dopamine. In Parkinson’s disease (PD) therapy, the inhibition of the oxidation of dopamine by MAO may elevate the levels of dopamine in the brain and prevent the generation of toxic by-products such as hydrogen peroxide. MAO-B inhibitors have found application as monotherapy in PD and it has been shown that MAO-B inhibitors may also be useful as adjuvants to L-dopa in PD therapy. For example, an earlier study has shown that the combination of L-dopa with (R)-deprenyl (a selective MAO-B inhibitor), may lead to a reduction of the dose of L-dopa required for alleviating the motor symptoms in PD patients. However, older MAO inhibitors may possess adverse side effects such as psychotoxicity, liver toxicity and cardiovascular effects. The irreversible mode of inhibition of the older MAO-B inhibitors, such as (R)-deprenyl, may also be considered as less desirable. After the use of irreversible inhibitors, it may require several weeks for the MAO enzyme to recover activity. In contrast, after administration of a reversible inhibitor, enzyme activity is recovered as soon as the inhibitor is cleared form the tissues. The adverse effects and disadvantages of the older MAO-B inhibitors prompted us to undertake the discovery of safer and reversible inhibitors of MAO-B. Such compounds may find application in the treatment of PD. Rationale It was recently discovered that (E)-8-(3-chlorostyryl)caffeine (CSC) is a potent inhibitor of MAO-B, with an IC50 value of 0.128 µM. CSC has a caffeine moiety, which is thought to be essential for MAO-B inhibition. It was also reported that a related series of 8- (phenoxymethyl)caffeine derivatives are potent and reversible inhibitors of MAO-A and –B. The IC50 values of the 8-(phenoxymethyl)caffeines ranged from 0.148–5.78 µM for the inhibition of MAO-B. For the purpose of this study the phenoxymethyl side-chain was replaced with a phenylsulfanyl moiety at C8. The aim of this study was therefore to synthesize a series of 8-[(phenylsulfanyl)methyl]caffeine analogues and to compare their MAO-B inhibition potencies to the previously synthesised 8-(phenoxymethyl)caffeine derivatives. A series of five 8-[(phenylsulfanyl)ethyl]caffeine analogues was also synthesized in order to determine the effect of carbon chain elongation on the potency of MAO inhibition. O C-8 N N O N N Caffeine Cl O N N (E) O N N CSC O N N O O N N 8-(Phenoxymethyl)caffeine O N N O N N S 8-[(Phenylsulfanyl)methyl]caffeine O N N S O N N 8-[(Phenylsulfanyl)ethyl]caffeine Compound R1 R2 1a H H 1b Cl H 1c Br H 1d F H 1e CH3 H 1f OCH3 H 1g OCH2CH3 H 1h H Cl 1i H Br Compound R1 R2 2a H H 2b Cl H 2c Br H 2d H Cl 2e H Br Methods The C8 substituted caffeine analogues were synthesised by reacting 1,3-dimethyl-5,6-diaminouracil with an appropriately substituted 2-(phenylsulfanyl)acetic acid or 3-(phenylsulfanyl)propanoic acid in the presence of a carbodiimide activating reagent, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC). Ring closure of the intermediary amide was effected by reaction with sodium hydroxide. Resulting theophylline analogues were subsequently methylated in the presence of iodomethane to yield the target compounds. The structures of the C8 substituted caffeine analogues were verified by NMR and MS analysis. The purities thereof were subsequently estimated by HPLC analysis. The 8-[(phenylsulfanyl)methyl]caffeine and 8-[(phenylsulfanyl)ethyl]caffeine analogues were evaluated as MAO-A and –B inhibitors. The recombinant human enzymes were used as enzyme sources. The inhibitory potencies of the caffeine derivatives were expressed as IC50 values (the concentration of a drug that is required for 50% inhibition in vitro). The time- dependency of inhibition of MAO-B by the most potent inhibitor was also evaluated in order to determine the reversibility of inhibition of the test compound. A study was also conducted to determine the inhibition mode of the most potent test compound, by constructing a set of Lineweaver Burk plots. Results The results showed that the 8-[(phenylsulfanyl)methyl]caffeine analogues were inhibitors of MAO-A and –B. The most potent inhibitor in the first series (1a–i) of this study were 8-[(3- bromophenylsulfanyl)methyl]caffeine and 8-[(4-bromophenylsulfanyl)methyl]caffeine with IC50 values of 4.90 and 4.05 µM, respectively. When these results were compared to those of the previously studied 8-(phenoxymethyl)caffeine derivatives it was found that, for these compounds, the bromine substituted homologues were also the most potent MAO-B inhibitors. The bromine substituted 8-(phenoxymethyl)caffeine derivatives exhibited IC50 values of 0.148 and 0.189 µM for those homologues containing bromine on the meta and para positions of the phenoxy side chain, respectively. In general, the 8- [(phenylsulfanyl)methyl]caffeine derivatives were found to be less potent MAO-B inhibitors than the 8-(phenoxymethyl)caffeine derivatives. The 8-[(phenylsulfanyl)methyl]caffeine derivatives also did not show as high a degree of selectivity for MAO-B (compared to MAO- A) as did the 8-(phenoxymethyl)caffeines. Similar to the 8-(phenoxymethyl)caffeines, the 8- [(phenylsulfanyl)methyl]caffeines also proved to be weak MAO-A inhibitors. The most potent inhibitor of MAO-A among the test compounds exhibited an IC50 value of 19.4 µM. The most potent MAO-A inhibitor among the previously studied 8-(phenoxymethyl)caffeines was more potent with an IC50 value of 4.59 µM. From these results it may be concluded that the phenoxy side chain is more suited for the design of caffeine derived MAO inhibitors than the phenylsulfanyl side chain. The results for the second series investigated in this study, the 8-[(phenylsulfanyl)ethyl]caffeines (2a–e), revealed the chlorine substituted derivatives to be the most potent MAO-B inhibitors. The meta and para chlorine substituted derivatives exhibited IC50 values of 5.67 and 7.79 µM, respectively, for the inhibition of MAO-B. Interestingly, the meta substituted derivative exhibited no inhibition toward the MAO-A isoenzyme. However, the 8-[(phenylsulfanyl)ethyl]caffeine derivatives were found to be very weak inhibitors of both MAO-A and –B and may be considered as less potent than the 8-[(phenylsulfanyl)methyl]caffeine derivatives. Since one of the aims of this study was to synthesise reversible MAO inhibitors, a time- dependency study was carried out with the best inhibitor (1i). The aim of this study was to determine the reversibility of inhibition by the 8-[(phenylsulfanyl)methyl]caffeine derivatives. From the results, it was concluded that the inhibition of MAO-B by compound 1i is reversible. To determine the mode of inhibition, a set of Lineweaver-Burk plots was constructed and since the plots were linear and intersected on the y-axis, it was concluded that 1i is a competitive inhibitor of MAO-B. Conclusion This study concludes that the phenoxymethyl side-chain is more suited for the design of caffeine derived MAO-B inhibitors than the (phenylsulfanyl)methyl side-chain. / Thesis (MSc (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2013.

Purpose Monoamine oxidase (MAO) consists of two isoforms, namely MAO-A and MAO-B. Both these isoforms are involved in the oxidation of dopamine. In Parkinson’s disease (PD) therapy, the inhibition of the oxidation of dopamine by MAO may elevate the levels of dopamine in the brain and prevent the generation of toxic by-products such as hydrogen peroxide. MAO-B inhibitors have found application as monotherapy in PD and it has been shown that MAO-B inhibitors may also be useful as adjuvants to L-dopa in PD therapy. For example, an earlier study has shown that the combination of L-dopa with (R)-deprenyl (a selective MAO-B inhibitor), may lead to a reduction of the dose of L-dopa required for alleviating the motor symptoms in PD patients. However, older MAO inhibitors may possess adverse side effects such as psychotoxicity, liver toxicity and cardiovascular effects. The irreversible mode of inhibition of the older MAO-B inhibitors, such as (R)-deprenyl, may also be considered as less desirable. After the use of irreversible inhibitors, it may require several weeks for the MAO enzyme to recover activity. In contrast, after administration of a reversible inhibitor, enzyme activity is recovered as soon as the inhibitor is cleared form the tissues. The adverse effects and disadvantages of the older MAO-B inhibitors prompted us to undertake the discovery of safer and reversible inhibitors of MAO-B. Such compounds may find application in the treatment of PD. Rationale It was recently discovered that (E)-8-(3-chlorostyryl)caffeine (CSC) is a potent inhibitor of MAO-B, with an IC50 value of 0.128 µM. CSC has a caffeine moiety, which is thought to be essential for MAO-B inhibition. It was also reported that a related series of 8- (phenoxymethyl)caffeine derivatives are potent and reversible inhibitors of MAO-A and –B. The IC50 values of the 8-(phenoxymethyl)caffeines ranged from 0.148–5.78 µM for the inhibition of MAO-B. For the purpose of this study the phenoxymethyl side-chain was replaced with a phenylsulfanyl moiety at C8. The aim of this study was therefore to synthesize a series of 8-[(phenylsulfanyl)methyl]caffeine analogues and to compare their MAO-B inhibition potencies to the previously synthesised 8-(phenoxymethyl)caffeine derivatives. A series of five 8-[(phenylsulfanyl)ethyl]caffeine analogues was also synthesized in order to determine the effect of carbon chain elongation on the potency of MAO inhibition. O C-8 N N O N N Caffeine Cl O N N (E) O N N CSC O N N O O N N 8-(Phenoxymethyl)caffeine O N N O N N S 8-[(Phenylsulfanyl)methyl]caffeine O N N S O N N 8-[(Phenylsulfanyl)ethyl]caffeine Compound R1 R2 1a H H 1b Cl H 1c Br H 1d F H 1e CH3 H 1f OCH3 H 1g OCH2CH3 H 1h H Cl 1i H Br Compound R1 R2 2a H H 2b Cl H 2c Br H 2d H Cl 2e H Br Methods The C8 substituted caffeine analogues were synthesised by reacting 1,3-dimethyl-5,6-diaminouracil with an appropriately substituted 2-(phenylsulfanyl)acetic acid or 3-(phenylsulfanyl)propanoic acid in the presence of a carbodiimide activating reagent, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC). Ring closure of the intermediary amide was effected by reaction with sodium hydroxide. Resulting theophylline analogues were subsequently methylated in the presence of iodomethane to yield the target compounds. The structures of the C8 substituted caffeine analogues were verified by NMR and MS analysis. The purities thereof were subsequently estimated by HPLC analysis. The 8-[(phenylsulfanyl)methyl]caffeine and 8-[(phenylsulfanyl)ethyl]caffeine analogues were evaluated as MAO-A and –B inhibitors. The recombinant human enzymes were used as enzyme sources. The inhibitory potencies of the caffeine derivatives were expressed as IC50 values (the concentration of a drug that is required for 50% inhibition in vitro). The time- dependency of inhibition of MAO-B by the most potent inhibitor was also evaluated in order to determine the reversibility of inhibition of the test compound. A study was also conducted to determine the inhibition mode of the most potent test compound, by constructing a set of Lineweaver Burk plots. Results The results showed that the 8-[(phenylsulfanyl)methyl]caffeine analogues were inhibitors of MAO-A and –B. The most potent inhibitor in the first series (1a–i) of this study were 8-[(3- bromophenylsulfanyl)methyl]caffeine and 8-[(4-bromophenylsulfanyl)methyl]caffeine with IC50 values of 4.90 and 4.05 µM, respectively. When these results were compared to those of the previously studied 8-(phenoxymethyl)caffeine derivatives it was found that, for these compounds, the bromine substituted homologues were also the most potent MAO-B inhibitors. The bromine substituted 8-(phenoxymethyl)caffeine derivatives exhibited IC50 values of 0.148 and 0.189 µM for those homologues containing bromine on the meta and para positions of the phenoxy side chain, respectively. In general, the 8- [(phenylsulfanyl)methyl]caffeine derivatives were found to be less potent MAO-B inhibitors than the 8-(phenoxymethyl)caffeine derivatives. The 8-[(phenylsulfanyl)methyl]caffeine derivatives also did not show as high a degree of selectivity for MAO-B (compared to MAO- A) as did the 8-(phenoxymethyl)caffeines. Similar to the 8-(phenoxymethyl)caffeines, the 8- [(phenylsulfanyl)methyl]caffeines also proved to be weak MAO-A inhibitors. The most potent inhibitor of MAO-A among the test compounds exhibited an IC50 value of 19.4 µM. The most potent MAO-A inhibitor among the previously studied 8-(phenoxymethyl)caffeines was more potent with an IC50 value of 4.59 µM. From these results it may be concluded that the phenoxy side chain is more suited for the design of caffeine derived MAO inhibitors than the phenylsulfanyl side chain. The results for the second series investigated in this study, the 8-[(phenylsulfanyl)ethyl]caffeines (2a–e), revealed the chlorine substituted derivatives to be the most potent MAO-B inhibitors. The meta and para chlorine substituted derivatives exhibited IC50 values of 5.67 and 7.79 µM, respectively, for the inhibition of MAO-B. Interestingly, the meta substituted derivative exhibited no inhibition toward the MAO-A isoenzyme. However, the 8-[(phenylsulfanyl)ethyl]caffeine derivatives were found to be very weak inhibitors of both MAO-A and –B and may be considered as less potent than the 8-[(phenylsulfanyl)methyl]caffeine derivatives. Since one of the aims of this study was to synthesise reversible MAO inhibitors, a time- dependency study was carried out with the best inhibitor (1i). The aim of this study was to determine the reversibility of inhibition by the 8-[(phenylsulfanyl)methyl]caffeine derivatives. From the results, it was concluded that the inhibition of MAO-B by compound 1i is reversible. To determine the mode of inhibition, a set of Lineweaver-Burk plots was constructed and since the plots were linear and intersected on the y-axis, it was concluded that 1i is a competitive inhibitor of MAO-B. Conclusion This study concludes that the phenoxymethyl side-chain is more suited for the design of caffeine derived MAO-B inhibitors than the (phenylsulfanyl)methyl side-chain. / Thesis (MSc (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2013.

Purpose: Monoamine oxidase (MAO) plays a key role in the treatment of Parkinson‟s disease (PD), since it is the major enzyme responsible for the catabolism of dopamine in the substantia nigra of the brain. Inhibition of MAO-B may conserve dopamine in the brain and provide symptomatic relief. The MAO-B inhibitors that are currently used for the treatment of PD, are associated with a variety of adverse effects (psychotoxic and cardiovascular effects) along with additional disadvantages such as irreversible inhibition of the enzyme. Irreversible inhibition may be considered a disadvantage, since following treatment with irreversible inhibitors, the rate by which the enzyme activity is recovered may be variable and may require several weeks. In contrast, following the administration of reversible inhibitors, enzyme activity is recovered when the inhibitor is cleared from the tissues. There exists therefore, a need to develop new reversible inhibitors of MAO-B which are considered to be safer than irreversible MAO-B inhibitors. Rationale: Recently discovered reversible MAO-B inhibitors include safinamide and (E)-8-(3-chlorostyryl)caffeine (CSC). Safinamide has a benzyloxy side chain, which is thought to be important for inhibition of MAO-B. CSC, on the other hand, consists of a caffeine moiety with a styryl substituent at C-8, which is also a critical feature for its inhibitory activity. In a previous study, the caffeine ring and the benzyloxy side chain were combined to produce a series of 8-benzyloxycaffeine analogues which proved to be potent new MAO-B inhibitors. In this study, caffeine was substituted with the phenoxymethyl functional group at C-8, instead of the benzyloxy moiety. The aim of this study was therefore to compare the MAO-B inhibition potencies of selected 8-(phenoxymethyl)caffeine analogues with the previously studied 8-benzyloxycaffeine analogues. In the current study, 8-(phenoxymethyl)caffeine (1) and nine 8-(phenoxymethyl)caffeine analogues (2-10) were synthesized and evaluated as inhibitors of recombinant human MAOA and –B. These analogues only differed in substitution on C3 and C4 of the phenoxymethyl phenyl ring. The substituents that were selected were halogens (Cl, F, and Br), the methyl group, the methoxy group and the trifluoromethyl group. These substituents are similar to those selected in a previous study where 8-benzyloxycaffeine analogues were evaluated as MAO inhibitors. This study therefore explores the effect that a variety of substituents on C3 and C4 of the phenoxymethyl phenyl ring will have on the MAO-A and –B inhibition potencies of 8-(phenoxymethyl)caffeine. Based on the results, additional 8-(phenoxymethyl)caffeine analogues with improved MAO-A and –B inhibition potencies will be proposed for investigation in future studies. Methods: The target, 8-(phenoxymethyl)caffeine, analogues were synthesized by reacting 1,3- dimethyl-5,6-diaminouracil with the appropriately substituted phenoxyacetic acid in the presence of a carbodiimide coupling agent. Ring closure was catalyzed in basic conditions and methylation of the resulting theophyline intermediates at C-7 was carried out with iodomethane. The structures and purities of all the target compounds were verified by NMR, MS and HPLC analysis. All of the 8-(phenoxymethyl)caffeine analogues were subsequently evaluated as MAO-A and –B inhibitors using the recombinant human enzymes. The inhibition potencies of the analogues were expressed as the IC50 values (concentration of the inhibitor that produces 50% inhibition). In addition, the time-dependency of inhibition of both MAO-A and –B was evaluated for two inhibitors in order to determine if these inhibitors interact reversibly or irreversibly with the MAO isozymes. A Hansch-type quantitative structure-activity relationship (QSAR) study was carried out in order to quantify the effect that different substituents on the phenyl ring of the 8-(phenoxymethyl)caffeine analogues have on MAO-B inhibition activity. Results: The results showed that among the test compounds, several analogues potently inhibited human MAO-B. The most potent inhibitor was 8-(3-bromophenoxymethyl)caffeine with an IC50 value of 0.148 μM toward human MAO-B. There were also inhibitors which displayed inhibition activities towards human MAO-A with IC50 values ranging from 4.59 μM to 34.0 μM. Compared to the 8-benzyloxycaffeine analogues, that were in general non-selective inhibitors, the 8-(phenoxymethyl)caffeine analogues, evaluated here, were selective for MAO-B. For example, 8-(3-bromophenoxymethyl)caffeine was found to be 141 fold more selective as an inhibitor of MAO-B than of MAO-A. Also, compared to the 8-benzyloxycaffeine analogues, the 8-(phenoxymethyl)caffeine analogues were slightly less potent MAO-B inhibitors. For example, 8-benzyloxycaffeine is reported to have an IC50 value of 1.77 μM for the inhibition of human MAO-B while 8-(phenoxymethyl)caffeine was found to have an IC50 value of 5.78 μM for the inhibition of human MAO-B. This study also shows that two selected analogues bind reversibly to MAO-A and –B, respectively, and that the mode of MAO-B inhibition is competitive for one representative compound. Qualitative inspection of the results revealed interesting structure-activity relationships. For the 8-(phenoxymethyl)caffeine analogues, bearing both the C3 and C4 substituents on the phenyl ring, the MAO-B activity significantly increases with halogen substitution. Furthermore, increased MAO-B inhibition was observed with increased electronegativity of the halogen substituent. To quantify these apparent relationships, a Hansch-type QSAR study was carried out. The results showed that the logarithm of the IC50 values (logIC50) correlated with Hansch lipophilicity (π) and the Swain-Lupton electronic (F) constants of the substituents at C-3 of the phenoxymethyl ring. The correlation exhibited an R2 value of 0.87 and a statistical F value of 13.6. From these results it may be concluded that electron-withdrawing substituents at C3 with a high degree of lipophilicity enhance MAO-B inhibition potency. These results are similar to those previously obtained for the series of 8-benzyloxycaffeine analogues. For this series, the MAO-B inhibition potencies correlated with the Hansch lipophilicity (π) and Hammett electronic (σ) constants of the substituents at C-3 of the benzyloxy ring. Similarly to the 8-(phenoxymethyl)caffeine analogues, electron-withdrawing substituents with a high degree of lipophilicity also enhance the MAO-B inhibition potencies of 8-benzyloxycaffeine analogues. / Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2011