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MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus Parkinson’s disease (PD) and Alzheimer’s disease (AD) are neurodegenerative diseases that significantly impact the quality of life of patients. The pathology of Parkinson’s disease is characterised by neuronal death of the nigrostriatal dopaminergic nerve terminals as well as the formation of Lewy bodies. The activity of monoamine oxidase (MAO) B increases in patients diagnosed with Parkinson’s diseases as they age resulting in oxidative stress, which in turn may lead to mitochondrial dysfunction as well as protein aggregation and misfolding. The pathology of Alzheimer’s disease includes the formation of amyloid plaques and neurofibrillary tangles (NFT). The aetiologies of these neurodegenerative diseases are still unknown. Current treatments are based on the presenting pathogenesis and symptoms of the respective diseases. Therefore, there is no treatment that stops or reverses neurodegeneration in Parkinson’s disease and Alzheimer’s disease and current treatment focuses on alleviating the symptoms. The MAO enzyme is involved in the pathogenesis of Parkinson’s disease and Alzheimer’s disease. MAO metabolises various neurotransmitters including noradrenaline (NA), dopamine (DA) and serotonin (5-HT). MAO is implicated in several neurodegenerative diseases due to the generation of reactive oxygen species (ROS) and participation in key pathological pathways. Monoamine oxidase type B (MAO-B) inhibition can be beneficial for the treatment of Parkinson’s diseases as well as Alzheimer’s disease. The current use of monoamine oxidase type A (MAO-A) inhibitors to treat depression is well documented. It is important to note that potent MAO-A inhibitors and serotonergic drugs should not be administered together as this can possibly induce serotonin toxicity. Caution should also be exercised with the use of potent irreversible inhibitors of MAO-A as they present with a higher occurrence of the “cheese reaction”. The dye compound methylene blue (MB), has shown promise as a therapeutic agent in studies conducted on Alzheimer’s disease and depression. Of particular interest is the ability of methylene blue to inhibit MAO. It was found that methylene blue as well as azure B, the major metabolite of methylene blue, potently inhibit MAO-A. It was also found that certain structural analogues of methylene blue also are good potency MAO-A inhibitors. For example, the dye compounds cresyl violet (IC50 = 0.0037 μM), Nile blue (IC50 = 0.0077 μM) and 1,9-dimethyl methylene blue (DMMB) (IC50 = 0.018 μM) are high potency MAO-A inhibitors. Due to the high potency MAO-A inhibition associated with these dye compounds, the present study investigated 22 commercially available dyes, that are similar in structure to methylene blue, as potential human MAO-A and MAO-B inhibitors. The inhibition potencies of the selected 22 dye compounds were determined by using recombinant human MAO-A and MAO-B enzymes. The IC50 values of the dyes were determined and were used as a measure of inhibition potency. Based on the IC50 values, acridine orange, oxazine 170 and Darrow red were identified as the highest potency inhibitors of this study. These compounds were subjected to further studies to determine the reversibility and mode of inhibition by means of dialysis and the construction of Lineweaver-Burk plots, respectively. It was found that acridine orange is a competitive and reversible inhibitor specific for MAO-A (IC50 = 0.017 μM). Oxazine 170 was identified as a competitive and reversible inhibitor specific for MAO-B (IC50 = 0.0065 μM). Darrow red exhibited competitive and reversible inhibition of MAO with specificity for neither of the isoforms (MAO-A, IC50 = 0.059 μM; MAO-B, IC50 = 0.065 μM). When compared to methylene blue (MAO-A, IC50 = 0.07 μM; MAO-B, IC50 = 4.37 μM), acridine orange and Darrow red exhibited more potent inhibition of MAO-A. The MAO-B inhibition potencies of oxazine 170 and Darrow red were also found to be higher than that of methylene blue. In conclusion, the MAO isoforms are implicated in the pathology of neurodegenerative and neuropsychiatric diseases such as Alzheimer’s disease, Parkinson’s disease and depression. Therefore, MAO is a viable and important target for possible medical intervention and drug treatments. The results of this study confirmed that some of the dye compounds evaluated in this study possess similar activity profiles to methylene blue. This can be explained by the structural similarity of the dye compounds with methylene blue. The dye compounds identified in this study can therefore be further investigated for possible preclinical development and be used as possible lead compounds to design future MAO inhibitors. Masters

PhD (Pharmacy with Pharmaceutical Chemistry), North-West University, Potchefstroom Campus Introduction: Human and animal health is constantly threatened by pathogenic microbes and the emergence of bacteria resistant to standard medicinal interventions has escalated the search for new and innovative pharmaceutical solutions. Natural medicines have received much attention in recent years as a potential answer to the problem of “super bugs”. Fulvic acid is a composition of organic acids found in nature and known for its anti-inflammatory, antimicrobial and antioxidant properties. Unfortunately, the detection of heavy metals embedded in the molecular structure of fulvic acids extracted from numerous environmental sources has rendered it unsafe for medicinal applications. A new invention by Fulhold Pharma Ltd to synthetically produce fulvic acid from sucrose, identified as Carbohydrate-Derived Fulvic Acid (CHD-FA), is a major international breakthrough in the production of a heavy metal free fulvic acid. CHD-FA is produced through a non-catalytic wet oxidation process and complies with standardised product specifications for molecular consistency and safety. CHD-FA has anti-inflammatory, antimicrobial and antioxidant therapeutic health benefits. Purpose: * To propose a theoretical model for the compound identified as the major constituent of CHD-FA. * The identification of the backbone structures embedded in CHD-FA. * To review the pharmacological properties of CHD-FA based on the composition of the backbone structures embedded in the molecular composition of CHD-FA’s cluster structure with the emphasis on anti-inflammatory, antibacterial, antifungal, antiviral and antioxidant properties. Methods: * A theoretical model, based on literature, was developed to describe the mechanisms involved in the non-catalytic wet oxidation process that has transformed sucrose into the major component (anhydrofulvic acid) of CHD-FA. Carbohydrate-Derived Fulvic Acid * Samples were collected for each of the different phases of the non-catalytic wet oxidation process, from the start-up of the reactor to the start of the exothermic reaction. Nuclear magnetic resonance spectroscopy (NMR) and liquid chromatography-tandem mass spectrometry (LC-MSMS) was used to analyse these samples to identify molecular changes during the various stages of this process. * Gas chromatography-mass spectrometry (GC-MS), Fourier’s transform infrared (FTIR) and NMR were used to provide general information on the mixture. * Matrix-assisted laser desorption and ionisation time-of-flight mass spectrometry (MALDI-TOF MS) was used to desorb and ionise CHD-FA without fragmentation, in order to identify and measure the absolute molecular weight of the main component in CHD-FA. * LC-MSMS was used to identify the most prominent backbone structures embedded in the CHD-FA molecular structure. These compounds were mainly identified by injecting a sample of CHD-FA in the LC-MSMS, identifying the major mass ions, generating empirical formulas associated with these peaks and then using software to predict molecular structures associated with these compounds. * The clinical applications associated with the anti-inflammatory, antimicrobial, antifungal, antiviral and antioxidant properties of CHD-FA were assessed through a comprehensive literature review of the characteristics of the backbone structures in the molecular structure of CHD-FA. Results: Objective 1: The detailed description of the theoretical pathway for the synthesis of the major component of CHD-FA, namely molecular fulvic acid, has provided evidence that the non-catalytic wet oxidation synthetic process of sucrose to produce molecular fulvic acid is a one-pot synthesis process consisting of a myriad of chemical reactions in the reactor. Colour changes in the reactor solution have confirmed the theoretical pathway description of a step-by-step process. The colours changed progressively from a light yellow to a dark brown colour. NMR and LC-MSMS analyses have confirmed that the colour changes demonstrated the transformation of sucrose into molecular fulvic acid. GCMS analysis revealed a concord between the structure of CHD-FA and penicillin-derivated fulvic acid. The MALDI-TOF MS identified 308 g/mol as the highest intensity peak with a natural abundance of 20.8 % in the spectrum and confirmed it as the most prominent component in CHD-FA. Batch-to-batch consistency of CHD-FA was recorded by chromatographic and spectroscopic data for more than 30 production runs over a four year period. Carbohydrate-Derived Fulvic Acid Objective 2: Similarities between the spectroscopic data of CHD-FA and literature data from environmental fulvic acids were indicated by FTIR and 13C NMR. However, CHD-FA has unique characteristics which differentiate it from environmental fulvic acids. CHD-FA has more carboxyl, ester, amide and aliphatic carbons in its molecular structure compared to the fulvic acid reference standards from the International Humic Substances Society. GC-MS confirmed the complexity of the molecular CHD-FA structure. The chromatogram overlay of CHD-FA and the reference standard, penicillin-derived fulvic acid (CAS 479-66-3), confirmed the presence of fulvic acid in CHD-FA. The most prominent component of the molecular structure of CHD-FA shown by LC-MSMS spectrum is 7,8‐dihydroxy‐3‐methyl‐10‐oxo‐1H,10H‐pyrano[4,3‐b]chromene‐9‐carboxylic acid with the empirical formulae of C14H10O7. This component is the dehydrated analogue of fulvic acid (C14H12O8) indicative of a loss of a water molecule during sample preparation. The LC-MSMS shows molecular ion m/z 290 and 24 prominent peaks, which represents the key structures in CHD-FA. It is evident that CHD-FA is a cluster of organic compounds. 24 prominent peaks were characterised as the backbone structures embedded in CHD-FA. This, with reference to the molecular composition of CHD-FA, is the most significant finding of the present study. Malic acid, maleic acid, levulinic acid, succinic acid, propenoic acid, phthalic acid, arabonic acid, itaconic acid, glucuronic acid, glutaric acid, benzene tri- and tetracarboxylic acids were identified as the backbone structures of CHD-FA. These backbone structures are interlinked with each other and with the parent structure via intermolecular bonding to form a cluster molecular structure. Objective 3: A comprehensive literature review of the clinical properties of the backbone structures of CHD-FA has demonstrated anti-inflammatory, antibacterial, antifungal, antiviral and antioxidant properties. These properties are therefore embedded in CHD-FA. Conclusion: CHD-FA, derived synthetically from sucrose through a non-catalytic wet oxidation process, is a pure form of fulvic acid. CHD-FA has the same medicinal properties as penicillin-derived reference standard fulvic acid and fulvic acids derived from various environmental sources. The identification of the twenty four backbone structures embedded in the supramolecular structure of CHD-FA is evidence that CHD-FA is a cluster of organic structures. This cluster of organic structures is responsible for the unique characteristics of CHD-FA, which include anti- Carbohydrate-Derived Fulvic Acid inflammatory, antimicrobial and antioxidant properties. The batch-to-batch consistency demonstrated for the manufacturing of CHD-FA in this study offers much potential for the use of CHD-FA as a natural pharmaceutical compound in the development of natural medicines. Doctoral

PhD (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus In the last few years, there has been an increase in the use of cannabis-based products for medicinal purposes. The preparation procedure, however, has not been standardised but is decided by the individual groups of scientists globally. As cannabis-based products are more frequently used for medicinal and recreational uses, standardised quality control methods must be developed, validated and harmonised. This study relates to the three major quality control parameters of interest in cannabis-based products in South Africa. These three quality control tests include cannabis potency, residual solvent residues and heavy metal residues. For cannabis potency a total of 840 samples were analysed in duplicate (1680 datapoints in total) and reported in an anonymous format. Samples were categorised into 7 different types: Edible, Extract, Infusion, Liquid, Other, Plant material, and Solid. Each category was divided into the following weight by percentage concentration levels: 1 wt.-%. The results indicated that high amounts of tetrahydrocannabinol (THC) are present in most of the cannabis-based products in South Africa. This is of concern due to the health implications of these products, and the current South African legislation related to cannabidiol (CBD) and THC. For heavy metal residues a total of 310 samples were analysed. The submitted samples were divided into different category classifications and grouped according to relevance for oral or inhalation specification. The results showed an alarming 15% sample failure rate compared to the oral specification limit and a 44% failure rate compared to the inhalation specification limit. It is of the utmost importance for manufacturers to have the appropriate quality control regimes in place, especially for heavy metal residues, in South Africa. Furthermore, it is imperative to ensure regulation is enforced and the South African public is educated about the risks associated with using these products. Solvent residue analysis employed a total of 279 samples in duplicate and the results are reported in an anonymised format. The results showed an alarming 37% sample solvent residue failure rate with respect to adherence to USP 467 specification. As seen from the facets discussed above, manufacturers, as well as cultivators should adhere to the current GMP guidelines set forth, to ensure the safety of the consumer as well as the quality of the product produced. With the current findings the safety of consumers is brought into question. With this data South African regulators as well as the public might be informed about the current state of cannabis-based products. Doctoral

PhD (Pharmacy), North-West University, Potchefstroom Campus Neurological disorders such as Alzheimer’s disease, Parkinson’s disease and schizophrenia are disorders that affect a large part of the population, especially the elderly. The cause of these disorders is still not completely known but a deficiency of certain neurotransmitters in the central nervous system is often responsible for the symptoms of theses disorders. For this reason, enzymes that metabolise neurotransmitters are considered as targets for the treatment of neurological diseases. The inhibition of these enzymes leads to increased levels of specific neuotransmitters and thus relief of the symptoms. Sometimes metabolic by-products contribute to the neurodegenerative processes, thus the inhibition of these enzymes may protect against neurodegeneration. The aim of this study was to evaluate the phenothiazine compound, methylene blue, and 31 dyes that are structuarally related to methylene blue as potential inhibitors of enzymes that are relevant to neurological disroders. Methylene blue interacts with various molecular targets and inhibits enzymes relevant to disease states such as depression and Alzheimer’s disease. In this study methylene blue and the related dyes were evaluated as inhibitors of the following enzymes: acethylcholinesterase (AChE), butyrylcholinesterase (BuChE), D-amino acid oxidase (DAAO), diamine oxidase (DAO), catechol-O-methyltransferase (COMT) and xanthine oxidase (XO). As mentioned, most of these enzymes are relevant to neurological disorders, with AChE and BuChE inhibitors already being used as treatment for Alzheimer’s disease. DAAO metabolises D-serine, and thus plays a role in glutaminergic neurotransmission in schizophrenia and Alzheimer’s disease. DAO metabolises histamine and primary amines and produces, in the same way as monoamine oxidases (MAO), harmful by-products during the catalytic cycle. COMT inhibitors are already established as treatment for Parkinson’s disease by increasing the levels of dopamine in the central nervous system. XO contributes to the formation of reactive oxygen species in the brain, which can lead to the destruction of neurons and thus contribute to the pathology of neurological diseases. The results show that various dyes are inhibitors of AChE and BuChE. Twelve and sixteen dyes were potent inhibitors of AChE and BuChE, respectively. Two of these dyes displayed sub-micromolar IC50 values for AChE and five for BuChE. The most significant inhibitor was acridine orange with an IC50 value of 2.18 μM and 0.22 μM towards AChE and BuChE, respectively. With DAAO, the dyes generally displayed good inhibition, with sixteen potent inhibitors and five inhibitors with sub-micromolar IC50 values. The two most potent inhibitors were hematoxylin and phenoxazine, which displayed IC50 values of 0.3 μM and 0.319 μM, respectively. Inhibition of DAO was less potent than the previous two enzymes, but there were nine dye compounds that exhibited potent inhibition. However, no compound had a sub-micromolar IC50 value. The two most potent inhibitors were darrow red and Nile blue, which displayed IC50 values of 2.33 μM and 2.45 μM, respectively. None of the dye compounds other than purpurin displayed significant inhibition of COMT. Purpurin displayed an IC50 value of 8.2 μM for the inhibition of COMT, and is therefore a moderately potent inhibitor. Purpurin possesses the catechol group as part of its structure, which confirms that compounds with catechol and nitro-catechol moieties often inhibit the COMT enzyme. For XO, a series of compounds from an in-house library were tested for inhibition. Most compounds did not inhibit XO with only six diplaying activity. Juglone and menadione displayed potent inhibition towards XO with IC50 values of 3.81 μM and 8.54 μM, respectively. Interestingly, four of the six compounds that displayed activity are naphthoquinones which represents a new class of XO inhibitors. 1,4-Naphthoquinone displayed an IC50 value of 29.3 μM. Nitrocatcehol compounds were also tested and were found to inhibit XO, with tolcapone displaying an IC50 value of 40.9 μM. This study shows that several of the dye compounds inhibit AChE, BuChE, DAAO and DAO. Methylene green, new methylene blue N and Nile blue are particularly noteworthy because these compounds possess IC50 values less than 10 μM for all these enzymes, and often have sub-micromolar IC50 values. The literature shows that these three dyes are also potent inhibitors of MAO. Thus, it can be concluded that certain dye compounds inhibit a wide variety of enzymes that are relevant to the pathology of neurological disorders. Doctoral

MSc (Pharmaceutical), North-West University, Potchefstroom Campus Parkinson’s disease (PD) is one of the leading causes of disability in the world. A better understanding of the aetiology of this disorder will lead to better medications, and would improve the quality of life for millions of patients worldwide. By inhibiting the degradation of dopamine in the midbrain, enzyme inhibitors of both catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO) are important drugs in the treatment of PD. Dopamine is degraded both peripherally and centrally by COMT to yield 3-O-methyldopa, which can reduce L-dopa absorption at the brain-blood barrier. COMT inhibitors have shown a great promise in reducing akinesia in controlled clinical trials. The MAO-catalysed degradation of dopamine yields reactive oxygen species which may accelerate neuronal degeneration in PD. Thus, MAO-B inhibitors are specifically used for PD pharmacotherapy. MAO-B inhibitors are considered to be safe medication with excellent safety profiles. By employing the hybrid theory for the design of new drugs, this study used the chalcone structure and the nitrocatechol moiety to design dual inhibitors for COMT and MAO. By using acid catalysed aldol condensation, aith a refluxtime of 24 – 26 hours, a series of nitrocatechol derivatives of chalcone was synthesised in good yields (71–84%). Bicyclic systems were also incorporated into the chalcone structure. An analysis of the structure-activity relationships showed an increase in MAO-B inhibition activity with the inclusion of either a methoxy or hydroxy group on the 5-position of the indanone bicyclic system. Overall the inhibition of MAO-B was moderate, with none of the compounds exhibiting IC50 values in the nanomolar range. The most potent IC50 for MAO-B inhibition was 7.26 μM for compound G, which bears chromanone as the bicyclic system. In contrast to their MAO inhibition potencies, the chalcones were good potency COMT inhibitors with IC50 values in the nanomolar range. The most potent COMT inhibitor was compound C with an IC50 value of 0.163 μM. The potency of this compounds can be attributed to the addition of the nitrocatechol moiety. This study concludes that the indanone bicyclic system substituted with methoxy or hydroxyl groups on the 5-position has potential for the design of dual inhibitors of MAO-B and COMT. Masters

MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus Flavonoids (different phytochemical compounds found in plants acting as phytoalexins), have been found to have potent antioxidant effects. This has led into studies conducted on its effect on the body in reducing oxidative stress in different diseases as they have the potential to scavenge free reactive oxidative species that causes harm to the body, causing degeneration of cells. In the search for medicines that can delay the progression of Alzheimer’s disease (AD), research into the use of flavonoids has become more popular based on the oxidative stress theory of AD. This has established its ability to reduce amyloid β plaque formation and tau protein formation in the different areas of the brain. This study aimed to develop a HPLC method for analysis of chrysin (flavone subgroup), kaempferol and quercetin (flavonol subgroup) after extraction from selected plant material (red and yellow onions and broccoli), identifying the flavonoid compounds, estimating their quantities and drawing comparisons between different processing and storage procedures. A rough screening method using TLC was also developed. A screening method was developed using TLC with two different mobile phases that proved adequate. This was a) toluene 80%: ethanol 20% and b) toluene 60%: ethyl acetate 30%: formic acid 10%. A HPLC method for analysis of chrysin, kaempferol and quercetin was developed and validated using the 10 mM phosphoric acid (H3PO4) 50%: methanol 25%: acetonitrile 25% as mobile phase with a flow rate set to 1 ml/min and the wavelength of detection set to 280 nm. A Kinetex® EVO C18 column (250 mm x 4.6 mm; 5 μm particle size and 100 Å pore size) was used. The plant material was processed to use whole, cut and blended plant material which were either fresh or frozen. Cooked plant material and grilled onions were also used for analysis of flavonoid content. Maceration, water bath extraction (WBE), ultrasonic extraction (USE) and microwave assisted extraction (MAE) were used to extract flavonoids from the prepared plant materials. Samples from these extractions were analysed on the HPLC. Comparisons were drawn between these different plant preparations, processing and extraction methods to establish the better method. In this study, best extraction results were achieved by MAE, followed by USE, maceration and lastly, WBE. A rough estimation of the chrysin, kaempferol and quercetin content led to the conclusion that insufficient concentrations of these flavonoids would be obtained from such small amounts of plant specimens used in this study. A more diverse diet with supplements of these compounds would be more beneficial to the AD patient, or any patient looking at natural treatment aimed at prevention. Masters

New N-acetyl/N-thiocarbamoylpyrazoline derivatives were designed and synthesized in high yields to assess their inhibitory activity and selectivity against human monoamine oxidase A and B. The most important chiral compounds were separated into their single enantiomers and tested. The impact of the substituents at N1, C3 and C5 positions as well the influence of the configuration of the C5 on the biological activity were analyzed. Bulky aromatic groups at C5 were not tolerated. p-Prenyloxyaryl moiety at C3 oriented the selectivity toward the B isoform. The results were also corroborated by molecular modelling studies providing new suggestions for the synthesis of privileged structures to serve as lead compounds for the treatment of mood disorders and neurodegenerative diseases.

Parkinson’s disease is characterised by the death of the nigrostriatal neurons and depletion of striatal dopamine. The standard symptomatic therapy consists of dopamine replacement with l -dopa, the metabolic precursor of dopamine, which represents the most effective treatment. Since monoamine oxidase (MAO) B is a key dopamine metabolising enzyme in the brain, MAO-B inhibitors are often used as adjuvants to l -dopa. In addition to the symptomatic benefits offered by MAO-B inhibitors, these drugs may also possess neuroprotective properties and possibly delay the progression of Parkinson’s disease. Based on the therapeutic use of MAO-B inhibitors, the present study evaluates a series of mono- and disubstituted derivatives of 4(3H)-quinazolinone as potential inhibitors of recombinant human MAO-A and MAO-B. Twelve C6-monosubstituted and nine N3/C6-disubstituted 4(3H)-quinazolinone derivatives were synthesised, which led to the discovery of novel quinazolinone derivatives with micromolar and submicromolar activities as inhibitors of MAO-B. The most potent mono- and disubstituted derivatives exhibited IC50 values of 6.35 μM (7f) and 0.685 μM (8b), respectively. This study identifies suitable substitution patterns for the design of 4(3H)-quinazolinone derivatives as MAO-B inhibitors.

The determination of catechol-O-methyltransferase (COMT) activity is considered valuable for various pharmaceutical and biomedical research projects. A specific high performance liquid chromatography-coulometric electrochemical detection method, for the assay of COMT activity was developed by measuring the formation of normetanephrine from norepinephrine. The chromatographic separation was achieved on a C18 reversed phase column with a mobile phase consisting of 10 mM sodium dihydrogen phosphate buffer, 4 mM sodium 1-octanesulfonate, 0.17 mM ethylenediaminetetra-acetic acid disodium salt, 6 % methanol and 4 % acetonitrile (pH ± 4.0). The detection of normetanephrine was achieved through electrochemical detection, with a coulometric cell potential setting of +450 mV. The flow rate was at 1 ml/min and the total run time was 45 min. The method was validated according to validation guidelines (Shabir 2006; European Medicines Agency 2011; US FDA 2018). The method was found to be linear (R2 > 0.99) over the analytical range (100 to 2500 ng/ml) for all the analytes. All the other validation parameters (sensitivity, precision, accuracy, recovery and stability) were acceptable and within range. The method was applied for the determination of COMT activity in rat liver homogenate test samples. The known selective COMT inhibitor entacapone was used as test inhibitor. The results confirmed the ability of entacapone to inhibit COMT activity by decreasing the production of all the metabolites of norepinephrine

WOS: 000481738100001, PubMed: 31422706, A series of benzo[b]thiophen-3-ols were synthesised and investigated as potential human monoamine oxidase (hMAO) inhibitors in vitro as well as ex vivo in rat cortex synaptosomes by means of evaluation of 3,4-dihydroxyphenylacetic acid/dopamine (DOPAC/DA) ratio and lactate dehydrogenase (LDH) activity. Most of these compounds possessed high selectivity for the MAO-B isoform and a discrete antioxidant and chelating potential. Molecular docking studies of all the compounds underscored potential binding site interactions suitable for MAO inhibition activity, and suggested structural requirements to further improve the activity of this scaffold by chemical modification of the aryl substituents. Starting from this heterocyclic nucleus, novel lead compounds for the treatment of neurodegenerative disease could be developed., Progetto di Ricerca Ateneo La Sapienza (Italy) [2014-C26A14AC5L]; POR FESR LAZIO 2014/2020 - REGIONE LAZIO - Avviso pubblico LIFE 2020; National Research Foundation of South AfricaNational Research Foundation - South Africa [85642, 96180], This work was supported by "Progetto di Ricerca Ateneo La Sapienza 2014-C26A14AC5L" (Italy) and POR FESR LAZIO 2014/2020 - REGIONE LAZIO - Avviso pubblico LIFE 2020 (Prof. Daniela Secci). The monoamine oxidase inhibition experiments were supported by grants from the National Research Foundation of South Africa (Grant specific unique reference numbers (UID) 85642, 96180). Opinions expressed and conclusions arrived at, are those of the authors and therefore the NRF do not accept any liability in regard thereto.

Series of structurally diverse 2-imidazoline derivatives have been synthesized by condensation of substituted aldehydes with ethylenediamine, Pd-catalyzed N-arylation of 2-imidazolines and by the formation of 1,2,4-oxadiazoles and benzoxazepines from 2-imidazoline-containing precursors. The 2-imidazoline derivatives were evaluated as potential inhibitors of human monoamine oxidase (MAO) A and B. Among the 2-imidazolines, good potency inhibitors were discovered with compound 9p (IC50 = 0.012 µM) being the most potent MAO-B inhibitor, while compound 9d (IC50 = 0.751 µM) was the most potent MAO-A inhibitor of the series. These potencies are in the same range as those of reference MAO inhibitors used in the clinic. Among 33 compounds evaluated, 13 exhibited IC50 values in the submicromolar range for the inhibition of an MAO isoform. It is postulated that the imidazoline moieties of some of these inhibitors may be recognized by the imidazoline I2-binding site of MAO. Good potency MAO inhibitors may be useful for the treatment of neuropsychiatric and neurodegenerative disorders such as depression and Parkinson’s disease, and future application for the treatment of prostate cancer, congestive heart failure and Alzheimer’s disease. In addition, high potency 2-imidazoline-derived MAO inhibitors may be used as potential probes for the imidazoline binding sites of the MAOs, as well as to determine alternative binding regions of imidazoline within the MAO active site.

In recent studies, we have investigated the monoamine oxidase (MAO) inhibition properties of pyrrolo[3,4-f]indole-5,7-dione and indole-5,6-dicarbonitrile derivatives. Since numerous high potency MAO inhibitors are present among these chemical classes, the present study synthesizes 44 additional derivatives in an attempt to further derive structure-activity relationships (SARs) and to establish optimal substitution patterns for MAO inhibition. The results show that, with the exception of one compound, all indole-5,6-dicarbonitrile derivatives (10) exhibit submicromolar IC50 values for the inhibition of MAO, with the most potent MAO-A inhibitor exhibiting an IC50 value of 0.006 μM while the most potent MAO-B inhibitor exhibits an IC50 value of 0.058 μM. Interestingly, an N-oxide derivative (4c) also proved to be a potent and nonspecific MAO inhibitor. With the exception of one compound, all of the pyrrolo[3,4-f]indole-5,7-diones (28) also exhibit submicromolar IC50 values for the inhibition of an MAO isoform. The most potent inhibitor exhibit an IC50 value of 0.011 μM for MAO-A. This study proposes that high potency MAO inhibitors such as those investigated here, may act as lead compounds for the development of treatments for neurodegenerative and neuropsychiatric disorders such as Parkinson's disease and depression.

The present study describes the synthesis of a series of 22 chalcone analogs. These compounds were evaluated as potential human MAO-A and MAO-B inhibitors. The compounds showed varied selectivity against the two isoforms. The IC50 values were found to be in the micromolar to submicromolar range. The Ki values of compound 16 were determined to be 0.047 and 0.020 μM for the inhibition of MAO-A and MAO-B, respectively. Dialysis of enzyme-inhibitor mixtures indicated a reversible competitive mode of inhibition. Most of the synthesized chalcone analogs showed a better selectivity toward MAO-B. However, introducing of 2,4,6-trimethoxy substituents on ring B shifted the selectivity toward MAO-A. In addition, we investigated the molecular mechanism of MAO-B inhibition by selected chalcone analogs. Our results revealed that these selected chalcone analogs increased dopamine levels in the rat hepatoma (H4IIE) cells and decreased the relative mRNA expression of the MAO-B enzyme

PhD (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus Parkinson's disease is a slowly progressive neurodegenerative disorder of unknown cause that selectively affects the dopaminergic, extrapyramidal nigrostriatal pathway. Parkinson's disease is a mid- or late life disease, presenting most often at ages 55-65, affecting 1-2% of the population over the age of 65. Current therapy is essentially symptomatic, and L-dopa, the direct precursor of dopamine, is the treatment of choice in more advanced stages of the disease. The oral bioavailability of L-dopa is estimated to be about 10% and less than 1% of the administered oral dose reaches the brain unchanged. In an attempt to overcome the problems with peripheral L-dopa metabolism, delivery difficulties and insufficient conversion of L-dopa to dopamine in the brain tissue, L-dopa prodrugs are proposed in this study. An L-dopa-lazabemide prodrug was thus proposed to overcome the problems associated with L-dopa absorption and delivery to the brain. Lazabemide, a monoamine oxidase (MAO) B inhibitor, slows depletion of dopamine stores and elevates dopamine levels produced by exogenously administered L-dopa. L-Dopa was linked at the carboxylate with the primary aminyl functional group of lazabemide via an amide, a strategy which is anticipated to protect L-dopa against peripheral decarboxylation and possibly also enhance the membrane permeability of the prodrug. Selected physicochemical and biochemical properties of the prodrug were determined. Although oral and i.p. treatment of mice with the prodrug did not result in an enhancement of striatal dopamine levels, DOPAC (Dihydroxyphenyl acetic acid) levels were significantly depressed compared to saline, L-dopa and carbidopa/L-dopa treatment. Secondly, in another attempt to overcome L-dopa's limited bioavailability and brain penetration, the present study synthesises four carrier-linked prodrugs of L-dopa in which 4-pyridylmethylamine, 2-(4-pyridyl)ethylamine, 2-(2-pyridyl)ethylamine and 3-phenyl-1-propylamine are linked to the carboxylate of L-dopa. Key physicochemical and biochemical parameters of the prodrugs were evaluated in an attempt to assess the potential of these prodrugs as vehicles to enhance the absorption and central delivery of L-dopa. Although the development of lazabemide has been discontinued, this compound is still used as reference MAO-B inhibitor in the in vitro screening of experimental MAO inhibitors. The third section of the present study aimed to characterise the in vitro MAO inhibition properties of lazabemide with respect to potency, isoform selectivity and reversibility. The results show that lazabemide is a selective inhibitor of human MAO-B with an IC50 value of 0.091 μM. For human MAO-A, lazabemide exhibits an IC50 of >100 μM. Interestingly, dialysis restores MAO-B activity only to a very small extent following inhibition by lazabemide, which shows that, in vitro inhibition persists and lazabemide may be viewed as an irreversible MAO-B inhibitor. Irreversible MAO inhibitors of the well-known propargylamine class include drugs that have been used clinically such as pargyline, selegiline and rasagiline, specifically for the treatment of depression and as adjuvants to L-dopa in Parkinson's disease. Due to their importance as MAO inhibitors, the fourth part of the present study synthesises a small series of novel propargylamine compounds that incorporate the pyridyl moiety. Pyridyl-derived propargylamines have not yet been investigated as potential MAO inhibitors. This study finds that the pyridyl-derived propargylamines do not inhibit either of the human MAO isoforms. Doctoral

New 4-(3-nitrophenyl)thiazol-2-ylhydrazone derivatives are proposed as dual-target-directed monoamine oxidase B (MAO-B) and acetylcholinesterase (AChE) inhibitors, as well as antioxidant agents, for the treatment of neurodegenerative disorders such as Parkinson's disease. Rational molecular design, target recognition and predicted pharmacokinetic properties have been evaluated by means of molecular modelling. Based on these properties, compounds were synthesized and evaluated in vitro as MAO-B and AChE inhibitors, and compared to the activities at their corresponding isozymes, monoamine oxidase A (MAO-A) and butyrylcholinesterase (BuChE), respectively. Anti-oxidant properties, potentially useful in the treatment of neurodegenerative disorders, have been also investigated in vitro . Among the evaluated compounds, three inhibitors may be considered as promising dual inhibitors of MAO-B and AChE, in vitro . MAO-B inhibition was also shown to be competitive and reversible for compound 19 .

MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus The possible treatment and prevention of Alzheimer’s disease (AD) is one of many clinical applications of methylene blue (MB) that has recently attracted much interest. Due to its ability to interact with various targets, MB exhibits multiple mechanisms by which the progression of neurodegenerative diseases may be decreased. Recently, it has been reported that azure B (AB), the major metabolite of MB, possesses superior effects at various pharmacological targets compared to MB. This finding raises the question that much of the documented pharmacological effects of MB observed in previous studies may in fact be due to the actions of AB. The structural similarities between MB and its metabolites have made the analysis of these compounds very challenging. The published analytical methods for MB and its metabolites have significant disadvantages such as low sensitivity, uneconomically high costs, the need of professionally trained personnel and very expensive, high technology apparatus. These disadvantages delays and limits the research into MB as a drug for the treatment of AD and other neurodegenerative disorders, and also hampers investigations into the pharmacology of MB. In this study, simple and cost effective analytical methods were developed to analyse and separate MB and its metabolites. An accurate, sensitive and reliable high performance liquid chromatography (HPLC) method with which MB and its metabolites were successfully separated, was developed and fully validated. A Synergi Polar-RP column (150 x 4.6 mm, 4 μ, 80 Å) and a mobile phase composed of two parts: ammoniumacetate that is dissolved in a mixture of water and methanol (part A) and a mixture of acetonitrile and methanol (part B). The analysis were done on a Hitachi Chromaster chromatographic system. Also, successful normal phase and reverse phase thin layer chromatography (TLC) methods were developed as a crude method for accessing the purity of MB. Masters

Introduction: The efficacy of L-dopa in the treatment of Parkinson’s disease depends on its metabolic conversion to dopamine in the brain, however extensive peripheral metabolism of L-dopa diminishes its availability for uptake into the brain. L-Dopa is extensively decarboxylated in the gastrointestinal tract and peripheral tissues by Aromatic L-Amino Acid Decarboxylase (AADC), and AADC inhibitors are thus frequently combined with L-dopa therapy. When AADC is inhibited, 3-Omethylation Catalysed by Catechol-O-Methyltransferase (COMT) becomes a dominant metabolic pathway for L-dopa, and COMT inhibitors may thus also be used as adjuncts to L-dopa in Parkinson’s disease. Monoamine Oxidase (MAO), in turn, metabolises dopamine in the brain, and MAO-B inhibitors may exert a dopamine sparing effect in the brain. Materials & Methods: Based on the roles of COMT and MAO in the metabolism of L-dopa and dopamine, the present study attempts to discover novel dual inhibitors of these enzymes. For this purpose, nitrocatechol derivatives of chalcone were synthesised and evaluated as inhibitors of COMT and MAO. The chalcone class of compounds is well known to potently inhibit MAO-B, while nitrocatechol derivatives (e.g. tolcapone and entacapone) are clinically used COMT inhibitors. Results: The results document that all of the derivatives are high potency in vitro inhibitors of rat liver COMT with IC50 values ranging from 0.07 to 0.29 μM. Under these experimental conditions, tolcapone and entacapone display IC50 values of 0.26 µM and 0.25 µM, respectively. The chalcones are less potent as inhibitors of MAO with the most potent inhibitor possessing a Ki of 4.6 µM for the in vitro inhibition of human MAO-B. Conclusion: This study shows that nitrocatechol derivatives of chalcone may act as COMT and MAO-B inhibitors, and proposes a general strategy for further enhancing MAO-B inhibition while retaining the potent COMT inhibition activity of this class

l-Dopa, the metabolic precursor of dopamine, is the treatment of choice for the symptomatic relief of the advanced stages of Parkinson's disease. The oral bioavailability of l-dopa, however, is only about 10% to 30%, and less than 1% of the oral dose is estimated to reach the brain unchanged. l-Dopa's physicochemical properties are responsible for its poor bioavailability, short half-life and the wide range of inter- and intrapatient variations of plasma levels. An l-dopa-lazabemide prodrug is proposed to overcome the problems associated with l-dopa absorption. Lazabemide is a monoamine oxidase (MAO)-B inhibitor, a class of compounds that slows the depletion of dopamine stores in Parkinson's disease and elevates dopamine levels produced by exogenously administered l-dopa. l-Dopa was linked at the carboxylate with the primary aminyl functional group of lazabemide via an amide, a strategy which is anticipated to protect l-dopa against peripheral decarboxylation and possibly also enhance the membrane permeability of the prodrug. Selected physicochemical and biochemical properties of the prodrug were determined and included lipophilicity (logD), solubility, passive diffusion permeability, pKa, chemical and metabolic stability as well as cytotoxicity. Although oral and i.p. treatment of mice with the prodrug did not result in enhanced striatal dopamine levels, 3,4-dihydroxyphenylacetic acid (DOPAC) levels were significantly depressed compared to saline, l-dopa and carbidopa/l-dopa treatment. Based on the results, further preclinical evaluation of the l-dopa-lazabemide prodrug should be undertaken with the aim of discovering prodrugs that may be advanced to the clinical stages of development.

This study examines a series of novel 3-benzyloxy-β-nitrostyrene analogues as a novel class of inhibitors of the monoamine oxidase (MAO) enzymes. MAO inhibitors are considered useful for the treatment of depression and Parkinson's disease, and have recently attracted attention as potential therapeutic agents for a range of disorders including Alzheimer's disease, prostate cancer and certain cardiomyopathies. This study shows that the 3-benzyloxy-β-nitrostyrene analogues are potent inhibitors of the MAO-B isoform with IC50 values in the nanomolar range (39–565 nM). Significantly, effectiveness towards MAO-B inhibition seems to be governed by the introduction of a 4″-fluoro-substituent on the benzyloxy ring, with compound 2b exhibiting the highest degree of MAO-B inhibition potency (IC50 = 0.039 μM) and selectivity (SI = 166) among the compounds investigated. Since some of the 3-benzyloxy-β-nitrostyrene analogues possess potencies that are comparable to that of the reversible inhibitor, safinamide (IC50 = 0.080 μM), it may be concluded that this class may be promising leads for the development of reversible and selective MAO-B inhibitors, that may be useful for the management of Parkinson's disease.

The monoamine oxidase (MAO) enzymes are of considerable pharmacological interest and inhibitors are used in the clinic for the treatment of major depressive disorder and Parkinson's disease. A limited number of studies have shown that the quinone class of compounds possesses MAO inhibition properties. Most notable among these is a report that 2,3,6-trimethyl-1,4-naphthoquinone (TMN), present in extracts of cured tobacco leafs, is a non-selective inhibitor of both MAO isoforms. An older study reports that 1,4-benzoquinone inhibits MAO-A and MAO-B from human synaptosomes. Both 1,4-naphthoquinones and 1,4-benzoquinone are reported to inhibit the MAOs with a reversible mode of action. Since the MAO inhibition properties of additional members of the 1,4-benzoquinone class of compounds have not yet been explored, the present study investigates a small series of four 1,4-benzoquinones which incorporate phenyl, benzyl, benzyloxy and cyclopentyl monosubstitution on C2. The 1,4-benzoquinones were found to be moderately potent MAO inhibitors with IC 50 values of 5.03–13.2 μM (MAO-A) and 3.69–23.2 μM (MAO-B). These values are comparable to those recorded for 1,4-benzoquinone of 4.82 μM (MAO-A) and 10.2 μM (MAO-B). Of interest however, is the finding that the 1,4-benzoquinones are irreversible inhibitors of MAO-A since prolonged incubation results in near complete inhibition, and enzyme activity is not recovered by dialysis. MAO-B is much less sensitive to inactivation by the 1,4-benzoquinones. These findings are discussed with reference to a possible mechanism by which irreversible inhibition occurs. It may be concluded that irreversible 1,4-benzoquinone-derived inhibitors may act as probes for investigating quinone reactive sites in the MAOs.

Methylene Blue (MB) is considered to have diverse medical applications and is a well-described treatment for methemoglobinemias and ifosfamide-induced encephalopathy. In recent years the focus has shifted to MB as an antimalarial agent and as a potential treatment for neurodegenerative disorders such as Alzheimer's disease. Of interest are reports that MB possesses antidepressant and anxiolytic activity in pre-clinical models and has shown promise in clinical trials for schizophrenia and bipolar disorder. MB is a noteworthy inhibitor of monoamine oxidase A (MAO-A), which is a well-established target for antidepressant action. MB is also recognized as a non-selective inhibitor of nitric oxide synthase (NOS) and guanylate cyclase. Dysfunction of the nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) cascade is strongly linked to the neurobiology of mood, anxiety and psychosis, while the inhibition of NOS and/or guanylate cyclase has been associated with an antidepressant response. This action of MB may contribute significantly to its psychotropic activity. However, these disorders are also characterised by mitochondrial dysfunction and redox imbalance. By acting as an alternative electron acceptor/donor MB restores mitochondrial function, improves neuronal energy production and inhibits the formation of superoxide, effects that also may contribute to its therapeutic activity. Using MB in depression co-morbid with neurodegenerative disorders, like Alzheimer's and Parkinson's disease, also represents a particularly relevant strategy. By considering their physicochemical and pharmacokinetic properties, analogues of MB may provide therapeutic potential as novel multi-target strategies in the treatment of depression. In addition, low MAO-A active analogues may provide equal or improved response with a lower risk of adverse effects.

The thionine dye, methylene blue (MB), is a potent inhibitor of monoamine oxidase (MAO) A, a property that may, at least in part, mediate its antidepressant effects in humans and animals. The central inhibition of MAO-A by MB has also been linked to serotonin toxicity (ST) which may arise when MB is used in combination with serotonergic drugs. Structural analogues and the principal metabolite of MB, azure B, have also been reported to inhibit the MAO enzymes, with all compounds exhibiting specificity for the MAO-A isoform. To expand on the structure-activity relationships (SARs) of MAO inhibition by MB analogues, the present study investigates the human MAO inhibition properties of five MB analogues: neutral red, Nile blue, new methylene blue, cresyl violet and 1,9-dimethyl methylene blue. Similar to MB, these analogues also are specific MAO-A inhibitors with cresyl violet (IC50 = 0.0037 μM), Nile blue (IC50 = 0.0077 μM) and 1,9-dimethyl methylene blue (IC50 = 0.018 μM) exhibiting higher potency inhibition compared to MB (IC50 = 0.07 μM). Nile blue also represents a potent MAO-B inhibitor with an IC50 value of 0.012 μM. From the results it may be concluded that non-thionine MB analogues (e.g. cresyl violet and Nile blue) also may exhibit potent MAO inhibition, a property which should be considered when using these compounds in pharmacological studies. Benzophenoxazines such as cresyl violet and Nile blue are, similar to phenothiazines (e.g. MB), representative of high potency MAO-A inhibitors with a potential risk of ST

Aim and objective Specific inhibitors of monoamine oxidase (MAO)-B are considered useful therapeutic agents in targeting neurological disorders like Alzheimer's and Parkinson's diseases. Due to the academic challenge of designing new hMAO-B inhibitors and the possibility of discovering compounds with improved properties compared to existing MAO-B inhibitors, a number of research groups are searching for new classes of chemical compounds that may act as selective hMAO-B inhibitors. Materials and methods Among these, chromone (4H-1-benzopyran-4-one) derivatives have recently emerged as a chemotype with specific and high potency MAO-B inhibition. Chromones are structurally related to a series of coumarins and chalcones, which are well-known inhibitors of MAO-B. Results The experimental evidence has demonstrated that most of the chromone skeleton derived compounds have shown potent, reversible and selective type of hMAO-B inhibitors. Conclusion The current review focuses on the MAO-B inhibitory properties of various synthetically derived chromones with specific emphasis on the structure-activity relationships and molecular recognition of MAO-B inhibition by this class. This review covers the recent updates present in the literature and will certainly provide a greater insight for the design and development of new class of potent chromone based selective MAO-B inhibitors.

Naturally occurring flavonoids display a plethora of different biological activities, but emerging evidence suggests that this class of compounds may also act as antidepressant agents endowed with multiple mechanisms of action in the central nervous system, increasing central neurotransmission, limiting the reabsorption of bioamines by synaptosomes, and modulating the neuroendocrine and GABAA systems. Due to their presence in foods, food-derived products, and nutraceuticals, we established their role and structure-activity relationships as reversible and competitive human monoamine oxidase (MAO) inhibitors. In addition, molecular modeling studies, which evaluated their modes of MAO inhibition, are presented. These findings could provide pivotal implications in the quest of novel drug-like compounds and for the establishment of harmful drug-dietary supplement interactions commonly reported in the therapy with antidepressant agents.

A screening study aimed at identifying inhibitors of the enzyme, monoamine oxidase (MAO), among clinically used drugs have indicated that the antirheumatic drug, leflunomide, is an inhibitor of both MAO isoforms. Leflunomide inhibits human MAO-A and MAO-B and exhibits IClt;subgt;50lt;/subgt; values of 19.1 μM and 13.7 μM, respectively. The corresponding Klt;subgt;ilt;/subgt; values are 17.7 μM (MAO-A) and 10.1 μM (MAO-B). Dialyses of mixtures of the MAO enzymes and leflunomide show that inhibition of the MAOs by leflunomide is reversible. The principal metabolite of leflunomide, teriflunomide (A77 1726), in contrast is not an MAO inhibitor. This study concludes that, although leflunomide is only moderately potent as an MAO inhibitor, isoxazole derivatives may represent a general class of MAO inhibitors and this heterocycle may find application in MAO inhibitor design. In this respect, MAO inhibitors are used in the clinic for the treatment of depressive illness and Parkinson's disease, and are under investigation as therapy for certain types of cancer, Alzheimer's disease and age-related impairment of cardiac function.

Lesetja J Legoabe,1 Anél Petzer,1 Jacobus P Petzer1,21Centre of Excellence for Pharmaceutical Sciences, 2Department of Pharmaceutical Chemistry, School of Pharmacy, North-West University, Potchefstroom, South AfricaAbstract: Based on a previous report that substituted 2-acetylphenols may be promising leads for the design of novel monoamine oxidase (MAO) inhibitors, a series of C5-substituted 2-acetylphenol analogs (15) and related compounds (two) were synthesized and evaluated as inhibitors of human MAO-A and MAO-B. Generally, the study compounds exhibited inhibitory activities against both MAO-A and MAO-B, with selectivity for the B isoform. Among the compounds evaluated, seven compounds exhibited IC50 values

In this study thirteen 2-aminopyrimidine derivatives were synthesised and screened as potential antagonists of adenosine A1 and A2A receptors in order to further investigate the structure activity relationships of this class of compounds. 4-(5-Methylfuran-2-yl)-6-[3-(piperidine-1-carbonyl)phenyl]pyrimidin-2-amine (8m) was identified as a compound with high affinities for both receptors, with an A2AKi value of 6.34 nM and an A1Ki value of 9.54 nM. The effect of selected compounds on the viability of cultured cells was assessed and preliminary results indicate low cytotoxicity. In vivo efficacy at A2A receptors was illustrated for compounds 8k and 8m since these compounds attenuated haloperidol-induced catalepsy in rats. A molecular docking study revealed that the interactions between the synthesised compounds and the adenosine A2A binding site most likely involve Phe168 and Asn253, interactions which are similar for structurally related adenosine A2A receptor antagonists North-West University, Medical Research Council (MRC) and the National Research Foundation (UID 76308)

The current pharmacological therapies for the treatment of Parkinson’s disease are mostly inadequate and recent, improved therapeutic agents are required. Two important molecular targets for the design of anti-parkinsonian therapeutic compounds are the adenosine A2A receptor and the enzyme, monoamine oxidase (MAO) B. Adenosine A2A receptor antagonists are a relatively new class of anti-parkinsonian agents, which act by potentiating dopamine-mediated neurotransmission via dopamine D2 receptors. MAO-B inhibitors are established therapy of Parkinson’s disease and inhibit the MAO-B-catalysed metabolism of dopamine in the brain. This conserves reduced dopamine stores and extends the action of dopamine. A2A antagonism and MAO-B inhibition have also been associated with neuroprotective effects, further establishing roles for these classes of compounds in Parkinson’s disease. Interestingly, caffeine, a known adenosine receptor antagonist, has been recently considered as a lead compound for the design and discovery of A2A antagonists and MAO-B inhibitors. This review summarizes the recent efforts to discover caffeinederived MAO-B inhibitors. The design of caffeine-derived A2A antagonists has been extensively reviewed previously. The prospect of discovering dual-target-directed compounds that act at both targets is also evaluated. Compounds that block the activation and function of both A2A receptors and MAO-B may have a synergistic effect in the treatment of patients with Parkinson’s disease

Studies have shown that natural and synthetic chalcones (1,3-diphenyl-2-propen-1-ones) possess monoamine oxidase (MAO) inhibition activities. Of particular importance to the present study is a report that a series of furanochalcones acts as MAO-B selective inhibitors. Since the effect of heterocyclic substitution, other than furan (and more recently thiophene, piperidine and quinoline) on the MAO inhibitory properties of the chalcone scaffold remains unexplored, the aim of this study was to synthesise and evaluate further heterocyclic chalcone analogues as inhibitors of the human MAOs. For this purpose, heterocyclic chalcone analogues that incorporate pyrrole, 5-methylthiophene, 5-chlorothiophene and 6-methoxypyridine substitution were examined. Seven of the nine synthesised compounds exhibited IC50 values

Methylene blue(MB) is reported to possess diverse pharmacological actions and is attracting increasing attention for the treatment of neurodegenerative disorders such as Alzheimer's disease. Among the pharmacological actions of MB, is the significant inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). These activities may, at least in part, underlie MB's beneficial effects in Alzheimer's disease. MB is metabolized to yield N-demethylated products of which azure B, the monodemethyl metabolite, is the predominant species. Azure B has been shown to be pharmacologically active and also possesses a variety of biological actions. Azure B therefore may contribute to the pharmacological profile of MB. Based on these considerations, the present study investigates the possibility that azure B may, similar to MB, act as an inhibitor of human AChE and BuChE. The results document that azure B inhibits AChE and BuChE with IC50 values of 0.486 μM and 1.99 μM, respectively. The results further show that azure B inhibits AChE and BuChE reversibly, and that the modes of inhibition are most likely competitive. Although the AChE and BuChE inhibitory activities of azure B are twofold and fivefold, respectively, less potent than those recorded for MB [IC50(AChE)=0.214 μM; IC50(BuChE)=0.389 μM] under identical conditions, azure B may be a contributor to MB's in vivo activation of the cholinergic system and beneficial effects in Alzheimer's disease. http://www.journals.elsevier.com/toxicology-and-applied-pharmacology/ This work is based on the research supported in part by the Medical Research Council and the National Research Foundation of South Africa (Grant specific unique reference numbers (UID) 85642 and 80647).

Aims: The phenothiazinium compound, methylene blue (MB), possesses diverse pharmacological actions and is attracting attention for the treatment of bipolar disorder and Alzheimer's disease. MB acts on both monoamine oxidase (MAO) and the nitric oxide (NO)-cGMP pathway, and possesses antidepressant activity in rodents. The goal of this study was to synthesise a structural analogue of MB, ethylthioniniumchloride (ETC), and to evaluate the effects of the structural changes on theMAO inhibitory and antidepressant properties of MB. This study also investigated the antidepressant properties of azure B, the major metabolite of MB, versus MB and imipramine as active comparators. Main methods: ETC and azure B were firstly evaluated as inhibitors of human MAO, and secondly for antidepressant-like activity in the acute forced swim test (FST) in rats, and compared to saline, imipramine and MB. Key findings: The results document that ETC is a reversible inhibitor of MAO-A and MAO-B with IC50 values of 0.510 μM and 0.592 μM, respectively, and that it is a weaker MAO-A inhibitor than MB and azure B. ETC and azure B were more effective than imipramine and MB in reversing immobility in the FST without inducing locomotor effects, with evidence supporting a serotonergic action. Of interest is the finding that ETC is more toxic for cultured cells than MB. Conclusion: Azure B may therefore be a contributor to the antidepressant effect of MB. Small structural changes made to MBretain its antidepressant effect, even though the resulting phenothiaziniumcompound possesses reduced MAO-A inhibitory potency http://www.journals.elsevier.com/life-sciences/ http://dx.doi.org/10.1016/j.lfs.2014.10.005 Medical Research Council and National Research Foundation of South Africa (Grant specific unique reference numbers (UID) 85642 and 80647)

Objectives Caffeine represents a useful scaffold for the design of monoamine oxidase (MAO) type B inhibitors. Specifically, substitution on the C8 position yields structures which are high-potency MAO-B inhibitors. To explore the structure–activity relationships of MAO-B inhibition by caffeine-derived compounds, this study examines the MAO inhibitory properties of a series of phenylalkylcaffeine analogues. Methods Employing the recombinant human enzymes, the potencies (IC50 values) by which the caffeine analogues inhibit MAO-A and MAO-B were measured. The reversibility of inhibition of a selected inhibitor was determined by measuring the recovery of enzyme activity after dilution and dialysis of enzyme-inhibitor mixtures. Key findings The results document that the phenylalkylcaffeine analogues are reversible and selective MAO-B inhibitors with a competitive mode of inhibition. The most potent analogue, 8-(7-phenylheptyl)caffeine, exhibits IC50 values for the inhibition of MAO-A and MAO-B of 3.01 μm and 0.086 μm, respectively. Increasing the length of the alkyl side chain leads to enhanced MAO-A and MAO-B inhibitory potency while introduction of a carbonyl group reduces MAO-B inhibitory potency. Conclusions Phenylalkylcaffeines represent a new class of high-potency MAO-B inhibitors with the longer alkyl side chains yielding enhanced inhibitory activity. Such compounds may represent useful leads for the development of anti-parkinsonian therapies.

Aims: Caffeine has been used as a scaffold for the design of inhibitors of monoamine oxidase (MAO) A and B. Substitution at the C8 position with a variety of moieties yields structures with high MAO inhibition potencies. Although theMAO inhibitory properties of numerous caffeine derivatives have been characterized, the possibility that caffeine inhibits the MAOs has not been investigated in detail. Based on the therapeutic applications and potential adverse effects of MAO inhibition, this study examines the interactions of caffeine with the MAOs. Main methods: Employing the recombinant human enzymes, the potencies by which caffeine inhibits the in vitro catalytic activities of theMAOs were recorded and expressed as the IC50 and Ki values. The reversibility of inhibition was determined by measuring the recovery of enzyme activity after dialysis of enzyme–caffeine mixtures. Key findings: Caffeine acts as a MAO inhibitor with Ki values of 0.70 mMand 3.83 mMfor the inhibition of MAO-A andMAO-B, respectively. The results showthat caffeine binds reversibly and competitively to bothMAO enzymes. Significance: Although structural modifications of caffeine lead to highly potent MAO inhibitors, caffeine is a weak inhibitor of MAO-A and MAO-B. At plasma concentrations (approximately 1–10 μM) achieved by normal human consumption, theMAO inhibitory potencies of caffeine are unlikely to be of pharmacological relevance in humans. The MAO inhibitory effects of caffeine should however be taken into consideration when using this drug in vitro and in tissue culture experiments where higher doses and concentrations of caffeine are often used. http://www.journals.elsevier.com/life-sciences/

In the present study, a series of 3,4-dihydro-2(1H)-quinolinone derivatives were synthesized and evaluated as inhibitors of recombinant human monoamine oxidase (MAO) A and B. The 3,4-dihydro-2(1H)- quinolinone derivatives are structurally related to a series of coumarin (1-benzopyran-2-one) derivatives which have been reported to act as MAO-B inhibitors. The results document that the quinolinones 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. 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 scaffold 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. It may be concluded that C7-substituted 3,4-dihydro-2(1H)-quinolinones are promising leads for the therapy of Parkinson’s disease. http://www.journals.elsevier.com/bioorganic-and-medicinal-chemistry-letters/

Monoamine oxidase (MAO) plays an essential role in the catabolism of neurotransmitter amines. The two isoforms of this enzyme, MAO-A and -B, are considered to be drug targets for the therapy of depression and neurodegenerative diseases, respectively. Based on a recent report that the phthalimide moiety may be a useful scaffold for the design of potent MAO-B inhibitors, the present study examines a series of 5-sulfanylphthalimide analogues as potential inhibitors of both human MAO isoforms. The results document that 5-sulfanylphthalimides are highly potent and selective MAO-B inhibitors with all of the examined compounds possessing IC50 values in the nanomolar range. The most potent inhibitor, 5-(benzylsulfanyl)phthalimide, exhibits an IC50 value of 0.0045 μM for the inhibition of MAO-B with a 427-fold selectivity for MAO-B compared to MAO-A. We conclude that 5-sulfanylphthalimides represent an interesting class of MAO-B inhibitors and may serve as lead compounds for the design of antiparkinsonian therapy. http://dx.doi.org/10.1016/j.bmcl.2012.08.113

A series of C7-substituted chromone (1-benzopyran-4-one) derivatives were synthesized and evaluated as inhibitors of recombinant human monoamine oxidase (MAO) A and B. The chromones are structurally related to a series of C7-functionalized coumarin (1-benzopyran-2-one) derivatives which has been reported to act as potent MAO inhibitors. The results of the current study document that the chromones are highly potent reversible inhibitors of MAO-B with IC50 values ranging from 0.008 to 0.370 μM. While the chromone derivatives also exhibit affinities for MAO-A, with IC50 values ranging from 0.495 to 8.03 μM, they are selective for the MAO-B isoform. Structure-activity relationships (SAR) show that 7-benzyloxy substitution of chromone is suitable for MAO-B inhibition with tolerance for a variety of substituents and substitution patterns on the benzyloxy ring. It may be concluded that 7-benzyloxychromones are appropriate lead compounds for the design of reversible and selective MAO-B inhibitors. With the aid of modeling studies, potential binding orientations and interactions of selected chromone derivatives in the MAO-A and -B active sites are examined. http://dx.doi.org/10.1016/j.bioorg.2012.08.003

Chromone has been reported to be a useful scaffold for the design of monoamine oxidase (MAO) inhibitors. In an attempt to discover highly potent MAO inhibitors and to contribute to the known structureeactivity relationships (SAR) of MAO inhibition by chromones, in the present study, we have synthesized a series of chromone derivatives substituted at C6 with a variety of alkyloxy substituents, and evaluated the resulting compounds as inhibitors of recombinant human MAO-A and -B. The results document that the C6-substituted chromones are potent reversible MAO-B inhibitors with IC50 values in the low nM range (2e76 nM). The chromones were also found to bind reversibly to MAO-A, but with lower affinities compared to MAO-B. It may therefore be concluded that C6-substituted chromones are highly potent MAO-B selective inhibitors and promising lead compounds for the development of therapy for neurodegenerative disorders such as Parkinson’s disease. The results of this study are discussed with reference to possible binding orientations of a selected C6-substituted chromone in the active site cavities of MAO-A and -B. http://dx.doi.org/10.1016/j.ejmech.2012.01.037

A previous study has shown that a series of C6-benzyloxy substituted chromones exhibit high binding affinities for human monoamine oxidase (MAO) B. In an attempt to discover additional chromones with potent and selective MAO-B inhibitory potencies and to further examine the structure-activity relationships of MAO-B inhibition by chromones, the series was expanded with homologues containing polar functional groups on C3 of the chromone ring. The results demonstrate that 6-[(3-bromobenzyl)oxy]chromones containing acidic and aldehydic functional groups on C3 act as potent reversible MAO-B inhibitors with IC50 values of 2.8 and 3.7 nM, respectively. Interestingly, a 2-hydroxy-2,3-dihydro-1-benzopyran-4-one derivative as well as open-ring 2-acetylphenol analogues of the chromones also were potent MAO-B inhibitors with IC50 values ranging from 4 to 11 nM. Chromone derivatives containing the benzyloxy substituent on C5 of the chromone ring, however, exhibit MAO-B inhibition potencies that are several orders of magnitude weaker. High potency inhibitors of MAO-B may find application in the therapy of neurodegenerative disorders such as Parkinson’s disease. http://dx.doi.org/10.1016/j.bmcl.2012.07.025

A recent study has reported that a series of 8-benzyloxycaffeines are potent and reversible inhibitors of both human monoamine oxidase (MAO) isoforms, MAO-A and -B. In an attempt to discover additional caffeine derivatives with potent MAO inhibitory activities, and to contribute to the known structure–activity relationships of MAO inhibition by caffeine derived compounds, the present study investigates the MAO inhibitory potencies of series of 8-phenoxymethylcaffeine and 8-[(phenylsulfanyl)methyl]caffeine derivatives. The results document that the 8-phenoxymethylcaffeine derivatives act as potent reversible inhibitors of MAO-B, with IC50 values ranging from 0.148 to 5.78 μM. In contrast, the 8-[(phenylsulfanyl)methyl]caffeine derivatives were found to be weak inhibitors of MAO-B, with IC50 values ranging from 4.05 to 124 μM. Neither the 8-phenoxymethylcaffeine nor the 8-[(phenylsulfanyl)methyl]caffeine derivatives exhibited high binding affinities for MAO-A. While less potent than the 8-benzyloxycaffeines as MAO-B inhibitors, this study concludes that 8-phenoxymethylcaffeines may act as useful leads for the design of MAO-B selective inhibitors. Such compounds may find application in the therapy of neurodegenerative disorders such as Parkinson’s disease. Using molecular docking experiments, this study also proposes possible binding orientations of selected caffeine derivatives in the active sites of MAO-A and -B. http://dx.doi.org/10.1016/j.bmc.2012.05.048

Methylene blue (MB) has been shown to act at multiple cellular and molecular targets and as a result possesses diverse medical applications. Among these is a high potency reversible inhibition of monoamine oxidase A (MAO-A) that may, at least in part, underlie its adverse effects but also its psycho- and neuromodulatory actions. MB is metabolized to yield N-demethylated products of which azure B, the monodemethyl species, is the major metabolite. Similar to MB, azure B also displays a variety of biological activities and may therefore contribute to the pharmacological profile of MB. Based on these observations, the present study examines the interactions of azure B with recombinant human MAO-A and -B. The results show that azure B is a potent MAO-A inhibitor (IC50=11 nM), approximately 6-fold more potent than is MB (IC50=70 nM) under identical conditions. Measurements of the time-dependency of inhibition suggest that the interaction of azure B with MAO-A is reversible. Azure B also reversibly inhibits the MAO-B isozyme with an IC50 value of 968 nM. These results suggest that azure B may be a hitherto under recognized contributor to the pharmacology and toxicology of MB by blocking central and peripheral MAO-A activity and as such needs to be considered during its use in humans and animals. http://www.journals.elsevier.com/toxicology-and-applied-pharmacology/ This work was supported by grants from the National Research Foundation and the Medical Research Council, South Africa.

In a recent study it was shown that 8-benzyloxycaffeine analogues act as potent reversible inhibitors of human monoamine oxidase (MAO) A and B. Although the benzyloxy side chain appears to be particularly favorable for enhancing the MAO inhibition potency of caffeine, a variety of other C8 oxy substituents of caffeine also lead to potent MAO inhibition. In an attempt to discover additional C8 substituents of caffeine that lead to potent MAO inhibition and to explore the importance of the ether oxygen for the MAO inhibition properties of C8 oxy-substituted caffeines, a series of 8-sulfanyl- and 8-aminocaffeine analogues were synthesized and their human MAO-A and -B inhibition potencies were compared to those of the 8-oxycaffeines. The results document that the sulfanylcaffeine analogues are reversible competitive MAO-B inhibitors with potencies comparable to those of the oxycaffeines. The most potent inhibitor, 8-{[(4-bromophenyl)methyl]sulfanyl}caffeine, exhibited an IC50 value of 0.167 lM towards MAO-B. While the sulfanylcaffeine analogues also exhibit affinities for MAO-A, they display in general a high degree of MAO-B selectivity. The aminocaffeine analogues, in contrast, proved to be weak MAO inhibitors with a number of analogues exhibiting no binding to the MAO-A and -B isozymes. The results of this study are discussed with reference to possible binding orientations of selected caffeine analogues within the active site cavities of MAO-A and -B. MAO-B selective sulfanylcaffeine derived inhibitors may act as lead compounds for the design of antiparkinsonian therapies. http://dx.doi.org/10.1016/j.bmc.2011.10.036

Published under the auspices of the French Société de Chimie Thérapeutique (SCT) A series of anilide derivatives were synthesized and evaluated as inhibitors of recombinant human monoamine oxidase (MAO) A and B. The most potent inhibitors among the derivatives that were initially evaluated were (2E)-N-(3-chlorophenyl)-3-phenylprop-2-enamide (2c) and (2E)-N-(3-bromophenyl)-3-phenylprop-2-enamide (2d) with IC50 values of 0.53 μM and 0.45 μM, respectively. These derivatives exhibited reversible and selective inhibition of MAO-B with binding affinities 37 fold higher for MAO-B than for MAO-A. Analysis of the possible binding interactions of these inhibitors with active site models of human MAO-A and –B led to the design of phenolic and benzonitrile derivatives of 2c and 2d. Among these were (2E)-N-(3-chlorophenyl)-3-(4-hydroxyphenyl)prop-2-enamide (7c) and (2E)-N-(3-bromophenyl)-3-(4-hydroxyphenyl)prop-2-enamide (7d) which inhibited MAO-B selectively and reversibly with IC50 values of 0.032 μM and 0.026 μM, respectively. These inhibitors were at least 14 fold more potent than 2c and 2d. This study concludes that N,3-diphenylprop-2-enamide is a suitable scaffold for the design of selective MAO-B inhibitors and structural modifications to enhance the binding affinities of the inhibitors for the MAO-B active site include substitution with halogens on the N-phenyl ring and substitution with hydroxyl and nitrile functional groups on the para and meta positions, respectively, of the C3 phenyl ring. Possible binding modes of these structures within the MAO-B active site are proposed with the emphasis on the interactions of the inhibitor halogens and the hydroxyl and nitrile functional groups with active site residues and water molecules.

The parkinsonian inducing agent, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is a cyclic tertiary allylamine exhibiting good monoamine oxidase B (MAO-B) substrate properties. MAO-B catalyzes the ring alpha-carbon 2-electron bioactivation of MPTP to yield the 1-methyl-4-phenyl-2,3-dihydropyridinium species (MPDP(+)). The corresponding 5-membered ring MPTP analogue, 1-methyl-3-phenyl-3-pyrroline, also undergoes MAO-B-catalyzed oxidation to give the 2-electron oxidation product, 1-methyl-3-phenylpyrrole. Here we report the kinetic deuterium isotope effects on V(max) and V(max)/K(m) for the steady-state oxidation of 1-methyl-3-phenyl-3-pyrroline and 1-methyl-3-(4-fluorophenyl)-3-pyrroline by baboon liver MAO-B, using the corresponding pyrroline-2,2,4,5,5-d(5) analogues as the deuterated substrates. The apparent isotope effects for the two substrates were 4.29 and 3.98 on V(max), while the isotope effects on V(max)/K(m) were found to be 5.71 and 3.37, respectively. The values reported for the oxidation of MPTP by bovine liver MAO-B with MPTP-6,6-d(2), as deuterated substrate, are (D)(V(max))=3.55; (D)(V(max)/K(m))=8.01. We conclude that the mechanism of the MAO-B-catalyzed oxidation of pyrrolinyl substrates is similar to that of the tetrahydropyridinyl substrates and that a carbon-hydrogen bond cleavage step is, at least partially, rate determining.