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

Biologically active peptides are important for many physiological functions in the human body and therefore serve as interesting starting points in drug discovery processes. In this work the neuropeptide substance P 1–7 (SP1–7, H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH), which has been demonstrated to reduce neuropathic pain and attenuate opioid withdrawal symptoms in animal models, has been addressed in a medicinal chemistry program with the overall aim of transforming this bioactive peptide into more drug-like compounds. Specific binding sites for this neuropeptide have been detected in the brain and the spinal cord. Interestingly, the smaller neuropeptide endomorphin-2 (EM-2, H-Tyr-Pro-Phe-Phe-NH2) also interacts with these binding sites, although 10-fold less efficient. In this work the structure–activity relationship of SP1–7 and EM-2, regarding their affinity to the SP1–7 binding site was elucidated using alanine scans, truncation, and terminal modifications. The C-terminal part of both peptides, and especially the C-terminal phenylalanine, was crucial for binding affinity. Moreover, the C-terminal functional group should preferably be a primary amide. The truncation studies finally resulted in the remarkable discovery of H-Phe-Phe-NH2 as an equally good binder as the heptapeptide SP1–7. This dipeptide amide served as a lead compound for further studies. In order to improve the drug-like properties and to find a plausible bioactive conformation, a set of rigidified and methylated dipeptides of different stereochemistry, and analogs with reduced peptide character, were synthesized and evaluated regarding binding, metabolic stability and absorption. Small SP1–7 analogs with retained affinity and substantially improved permeability and metabolic stability were identified. Beside peptide chemistry the synthetic work included the development of a fast and convenient microwave-assisted protocol for direct arylation of imidazoles. Furthermore, microwave-assisted aminocarbonylation using Mo(CO)6 as a solid carbon monoxide source was investigated in the synthesis of MAP amides and for coupling of imidazoles with amino acids. In a future perspective the present findings, together with the fact that some of the SP1–7 analogs discovered herein have been shown to reproduce the biological effects of SP1-7 in animal studies related to neuropathic pain and opioid dependence, can ultimately have an impact on drug discovery in these two areas.

Made available in DSpace on 2015-05-14T13:21:44Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 4637140 bytes, checksum: f9c50e9a2115f5a805442d163ed54f1e (MD5) Previous issue date: 2012-12-14 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The Morita-Baylis-Hillman Adducts (MBHA) is a class of molecules studied by our research group on synthetic, theoretical and bioactivity aspects. In this work, we present Quantitative Structure-Activity Relationship (QSAR) models involving 32 aromatic MBHA. Initially, the most stable conformations of thirty-two MBHA were investigated by theoretical methods, which were used to construct models. For this study, were obtained potential energy curves using AM1 semi-empirical method, considering rotational degrees of freedom (sigma bonds). From these curves, the less energy conformation to each molecule was selected and optimized at B3LYP/6- 31+G(d) level, considering solvent effects through Polarizable Continuum Model (PCM). Proton Nuclear Magnetic Ressonance data are in agreement with the conformational study. Intramolecular Hydrogen Bonds (IHB) are presents in the most of the studied compounds, according to structural characterization and QTAIM calculations. Curiously, compounds that showed hydrogen bonds involving the nitro and hydroxyl groups have the best values of biological activity (IC50). An explanation is based on redox mechanism of action of nitrocompounds. NBO (Natural Bond Orbital) charges and LUKO (Lowest Unoccupied Kohn-Sham Orbitals) analysis at the ortho-nitro group are in agreement with these analyses. Considering quantum calculations and structural observations, four descriptors were selected a priori and submitted to a QSAR study using PLS (Partial Least Squares) and MLR (Multiple Linear Regression) modeling. A second QSAR approach was made from the another set of descriptors obtained through the online platform E-DRAGON, which were submitted to a variable selection method. The quality parameters obtained for models indicate that both are robust and predictive. / Os Adutos de Morita-Baylis-Hillman (AMBH) compreendem uma classe de moléculas investigadas por nosso grupo de pesquisas nos aspectos sintéticos, teóricos e de bioatividade. Neste trabalho, apresentamos modelos de Relação Quantitativa entre a Estrutura Química e a Atividade Leishmanicida (QSAR) envolvendo 32 AMBH aromáticos. Deste modo, inicialmente foram investigadas as conformações mais estáveis de cada composto através de métodos teóricos, as quais foram utilizadas na construção dos modelos. Foram obtidas curvas de energia potencial utilizando o método semi-empírico AM1, considerando graus de liberdade rotacionais (ligações sigma). A partir destas curvas, a conformação de menor energia para cada molécula foi selecionada e otimizada ao nível B3LYP/6-31+G(d), considerando os efeitos do solvente aquoso usando PCM ( Polarizable Continuum Model ). Dados espectroscópicos de Ressonância Magnética Nuclear de prótons corroboraram o estudo conformacional. Ligações de Hidrogênio Intramoleculares (LHI) se mostraram presentes na maioria das moléculas estudadas, conforme caracterização estrutural e cálculos QTAIM ( Quantum Theory Atoms in Molecules ). Curiosamente, os compostos que apresentaram Ligações de Hidrogênio envolvendo o grupo nitro (NO2) e a hidroxila (OH) possuem melhores valores de atividade biológica (menor IC50). Uma explicação está baseada no mecanismo de ação redox de nitrocompostos. Observação das cargas NBO ( Natural Bond Orbitals ) e análise dos orbitais de fronteira LUKO ( Lowest Unoccupied Kohn-Sham Orbitals ) ao nível do grupo orto-nitro estão de acordo com estas análises. Considerando os cálculos quânticos, bem como observações estruturais, quatro descritores foram selecionados a priori e submetidos a um estudo QSAR ( Quantitative Structure- Activity Relationships ) utilizando modelagem PLS ( Partial Least Squares ) e MLR ( Multiple Linear Regression ). Uma segunda abordagem QSAR foi realizada a partir de outro conjunto de descritores obtidos através da plataforma online E-DRAGON, os quais foram submetidos ao método de seleção de variáveis OPS ( Ordered Predictor Selection ). Os parâmetros de qualidade obtidos para os modelos indicam que ambos são robustos e preditivos.

As estruturas e propriedades biológicas das amidas piplartina e a piperina, isoladas respectivamente de Piper tuberculatum e P. nigrum, inspiraram a síntese de 89 derivados e 7 esters estruturalmente relacionadas. As preparações envolveram metodologias tradicionais e os compostos purificados tiveram suas estruturas caracterizadas por análises espectroscópicas e espectrométricas. Os estudos de fragmentação por IE e IES indicaram a clivagem preferencial da ligação N-CO no caso das cinamamidas, dienamidas e cinamimidas. Estudos computacionais envolvendo afinidade protônica e energias de ligação confirmaram a fragmentação preferencial da ligação amídica para as amidas. A citotoxicidade de 89 substâncias foi avaliada contra três células leucêmicas (K562, Nalm6 e Raji) e a partir dos valores de IC50 foram realizados estudos de relação estrutura-atividade (SAR). As linhagens K562 e a Nalm6 foram a mais resistente e vulnerável, respectivamente, e as amidas piplartina (1a), N-Ciclohexil-N-(ciclohexilcarbamoil)-3-(3,4,5-trimetoxifenil)propanamida (1n), e (E)-N,N-dibutil-3-(3,4-dimetoxifenil)acrilamida (13h) foram as mais ativas com IC50 de 0,34 µM; 0,84 µM e 1,88 µM contra K562 e (E)-N-ciclohexil-N-(ciclohexilcarbamoil)-3-(3,4-dimetoxifenil)acrylamida (13i) com IC50 de 0,98 µM contra Nalm6. A avaliação de atividade leishmanicida de 18 substâncias não se mostrou promissora. As abordagens qualitativas e quantitativas foram feitas baseadas nos descritores moleculares gerados pelo programa VolSurf+. A partir de métodos quimiométricos tais com PLS, algoritmo genético, árvores de decisão foi possível gerar modelos para correlacionar às propriedades moleculares com a atividade biológica. As propriedades de absorção, distribuição, metabolismo e excreção e os equilíbrios entres as regiões hidrofílicas e hidrofóbicas foram importantes para atividade citotóxica. O estudo de ancoragem molecular mostrou que as amidas (E)-N,N-dibutil-3-(3,4,5-trimetoxifenil)acrilamida (1l), 1n, (E)-3-(4-clorofenil)-N-ciclohexil-N-(ciclohexilcarbamoil)acrilamida (5a), 13h e 13i podem atuar como inibidores das histonas desacetilases particularmente HDAC4 e HDAC8. / The structures and biological properties of the amides piplartine and piperine isolated from Piper tuberculatum and P. nigrum respectively, inspired the synthesis of derivatives 89 and 7 esters structurally related. Their preparations were achieved using classical procedures and the purified amides were submitted to spectroscopic and spectrometric characterization. The study of fragmentation process by EI and ESI suggested the preferential cleavage of the N-CO bond of cinnamamides, dienamides and cinnamimides. The cytotoxicity of 89 compounds was evaluated against three leukemic cells (K562, Nalm6 and Raji) and based on IC50 values the structure-activity relationship (SAR) was performed. While the K562 and Nalm6 cells were the more resistant and more sensitive, respectively, the amides piplartine (1a), N-cyclohexyl-N-(ciclohexylcarbamoyl)-3-(3,4,5-trimethoxyphenyl)propanamide (1n) and (E)-N,N-dibutyl-3-(3,4-dimethoxyphenyl)acrylamide (13h) were in general the most active with IC50 of 0.34 µM, 0.84 µM and 1.88 µM against K562 and (E)-N-cyclohexyl-N-(ciclohexylcarbamoyl)-3-(3,4-dimethoxyphenyl)acrylamide (13i) with IC50 of 0.98 µM against Nalm6. The evaluation of leishmanicidal activity of 18 substances was also performed but was not promising. Qualitative and quantitative approaches were made based on molecular descriptors generated by VolSurf+ program. The chemometric methods such as PLS, genetic algorithm, decision trees generated models to correlate molecular properties with the biological activity. The absorption, distribution, metabolism and excretion properties and a balance between hydrophilic and hydrophobic moieties of the amides were important for an optimized activity. The molecular docking revealed that amides such as (E)-N,N-dibutyl-3-(3,4,5-trimethoxyphenyl)acrylamide (1l), 1n, (E)-3-(4-chlorophenyl)-N-cyclohexil-N-(cyclohexylcarbamoyl)acrylamide (5a), 13h and 13i have potential to act as possible inhibitors of histone deacetylase proteins particularly HDAC4 and HDAC8.

Environ un tiers des cancers est dû à la dérégulation du facteur de transcription c-Myc. Les mécanismes par lesquels ce facteur de transcription est impliqué dans le processus de cancérogenèse commencent à être mieux compris grâce notamment à l'identification de ses gènes cibles. Parmi eux a été identifié, à la fin des années 2000, le gène dnph1 codant pour une protéine surexprimée dans de nombreux cancers. Cependant, à ce jour, le rôle et la fonction biologiques de cette nouvelle cible thérapeutique restent méconnus. Cette protéine a été caractérisée au laboratoire comme étant une 2'-désoxyribonucléoside 5'-monophosphate N-hydrolase, activité jamais décrite jusqu'alors. Si les 2'-désoxyribonucléosides 5'-monophosphates sont substrats de cette enzyme, il a été montré que les ribonucléotides puriques canoniques sont des inhibiteurs compétitifs. Au cours de ces travaux de thèse, nous avons entrepris différentes études de relation structure-activité autour d'analogues ribonucléosidiques 5'-monophosphates dans le but d'identifier des inhibiteurs plus affins. Une première famille d’analogues modifiés en position 6 de la purine par différents groupements de taille et fonction variables a été synthétisée et les premiers inhibiteurs micromolaires de DNPH1 ont ainsi pu être identifiés. La co-cristallisation de certains composés avec l'enzyme a guidé la conception d'inhibiteurs de deuxième génération, modifiés en positions 6 et 2. Un léger effet additif sur les constantes d’inhibition a alors été observé et des inhibiteurs sub-micromolaires ont été identifiés. Des tests in vitro sur des lignées cellulaires cancéreuses surexprimant DNPH1 ont montré une activité cytotoxique micromolaire de certains des composés synthétisés. D'autres modifications portant sur la partie ribose-phosphate et le squelette purique ont été abordées dans le but de renforcer l'affinité et la stabilité biologique des inhibiteurs. L'ensemble de ces travaux a permis d'une part de mieux caractériser cette nouvelle cible et d'autre part d'identifier les premiers inhibiteurs micromolaires cytotoxiques. Ces résultats ouvrent des perspectives pour la conception de molécules plus efficaces. / About one third of the cancers is due to the deregulation of the transcription factor c-Myc. The gene dnph1 was identified a decade ago as a target gene of the c-Myc oncoprotein and encodes for a protein which is frequently over-expressed in several cancers. However, its biological role remains still unknown. It was recently shown that DNPH1 is a novel 2'-deoxyribonucleoside 5'-monophosphate N-hydrolase and that natural purine ribonucleotides act as competitive inhibitors. The aim of this thesis was the synthesis of strong inhibitors in order to study this new potential cancer target DNPH1. Several structure-activity relationships were built around ribonucleoside 5'-monophosphate derivatives. A first series of compounds modified at the 6 position of the purine core has been synthesized and enabled us to identify the first micromolar inhibitiors. Thanks to these inhibitors, X-ray structures of DNPH1 in interaction with inhibitors have been resolved and led us to the development of a new generation of analogues modified at the 6 and 2 positions. A slight additional effect on the inhibitory potency was noticed and some sub-micromolar inhibitors were identified. Among the synthesized compounds, several have shown micromolar cytotoxic effects against human cancer cells over-expressing DNPH1. Other modifications on the ribose-phosphate and the purine moieties have also been considered in order to increase both biological stability and affinity of the inhibitors. This work allowed a better characterization of the enzyme active site as well as the identification of new cytotoxic compounds. These results pave the way for the design of more potent inhibitors.

The herein presented Ph.D. dissertation describes kinetic and structural characterization of human glutamate carboxypeptidases II and III (GCPII and GCPIII) using a complete panel of their natural substrates. These enzymes hydrolyze C-terminal glutamate from their substrates. They share 67 % sequence identity and also similar enzymatic activities. This thesis quantitatively compares human GCPII and GCPIII in terms of their ability to hydrolyze the substrates N-acetyl-L-aspartyl-L-glutamate (NAAG), folyl-poly-γ-L-glutamic acids (FolGlun) and β-citryl-L-glutamate (BCG). We demonstrated that GCPIII hydrolyzes its substrates in a metal- dependent manner, that BCG is a specific substrate of GCPIII, and that NAAG and FolGlun are specific substrates of GCPII. We also provide indirect biochemical evidence that GCPIII might feature a heterometallic active-site cluster. Additionally, we characterized the relevance of a surface exosite of GCPII, the arene-binding site (ABS), for the hydrolysis of FolGlun substrates using mutagenesis and enzyme kinetics and showed that polymorphic His475Tyr variant of GCPII hydrolyzes FolGlun substrates with the same kinetic parameters as the wild-type enzyme. Furthermore, this thesis focuses on structural aspects of the substrate specificities of GCPII and GCPIII: we present...

Polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) and polychlorinated biphenyls (PCBs) are omnipresent and persistent environmental pollutants. In particular, 29 congeners are of special concern, and these are usually referred to as dioxin-like compounds (DLCs). In the European Union, the risks associated with DLCs in food products are estimated by a weighted sum of the DLCs’ concentrations. These weights, also called toxic equivalency factors (TEFs), compare the DLCs’ potencies to the most toxic congener, 2,3,7,8-tetrachloro-dibenzo-p-dioxin (2378- TCDD). The toxicological effects of PCDD/Fs and PCBs are diverse, ranging from chloracne and immunological effects in humans to severe weight loss, thymic atrophy, hepatotoxicity, immunotoxicity, endocrine disruption, and carcinogenesis in rodents. Here, the molecular structures of DLCs were used as the basis to study the congeneric differences in in vitro data from both human and rodent cell responses related to the aryl hydrocarbon receptor (AhR). Based on molecular orbital densities and partial charges, we developed new ways to describe DLCs, which proved to be useful in quantitative structure-activity relationship modeling. This thesis also provides a new approach, the calculation of the consensus toxicity factor (CTF), to condense information from a battery of screening tests. The current TEFs used to estimate the risk of DLCs in food are primarily based on in vivo information from rat and mouse experiments. Our CTFs, based on human cell responses, show clear differences compared to the current TEFs. For instance, the CTF of 23478-PeCDF is as high as the CTF for 2378-TCDD, and the CTF of PCB 126 is 30 times lower than the corresponding TEF. Both of these DLCs are common congeners in fish in the Baltic Sea. Due to the severe effects of DLCs and their impact on environmental and human health, it is crucial to determine if other compounds have similar effects. To find such compounds, we developed a virtual screening protocol and applied it to a set of 6,445 industrial chemicals. This protocol included a presumed 3D representation of AhR and the structural and chemical properties of known AhR ligands. This screening resulted in a priority list of 28 chemicals that we identified as potential AhR ligands.

This thesis is engaged in the synthesis of polysubstituted pyrimidines with anti- inflammatory properties. Such molecules can inhibit production of prostaglandin E2 (PGE2). The aim of this study was to enhance water-solubility and anti-inflammatory efficacy of such derivatives via structural modifications of the lead scaffold. Among applied synthetic tools, the Suzuki-Miyaura cross-coupling was the prevalent reaction, however, many other synthetic procedures (Heck reaction, condensation, borylation, ozonolysis, nucleophilic substitution, etc.) were utilized as well. Overall, 43 final products were prepared. The anti-inflammatory efficacy (inhibition of PGE2 production) was successfully increased as the most potent compound achieved three orders of magnitude higher activity compared to the current lead structure WQE-134. Furthermore, no general influence of the length of the substituent in the C5 position of pyrimidine (C5pyr) on the anti-inflammatory efficacy of synthesized compounds was observed. Significant bioavailability obstacle in future development of the current lead WQE-134 is its poor solubility which was successfully enhanced by introduction of heteroatom bearing moieties to C5pyr. The most water-soluble compound achieved two orders of magnitude higher solubility than WQE-134 while...

Le carbonate de diméthyle (DMC) est utilisé comme intermédiaire en chimie des polymères. Actuellement, la production industrielle du DMC met en jeu un procédé polluant et dangereux. En revanche, la synthèse du DMC à partir de méthanol et de CO2 est un procédé « vert ». Cependant, la thermodynamique de la réaction est très défavorable, se traduisant par une conversion inférieure à 1%. L'objectif de cette thèse est de développer et d'étudier des catalyseurs très actifs qui, couplés à un réacteur membranaire, permettraient d'augmenter le rendement et l'activité. Ce manuscrit couvre plusieurs aspects : i) une étude de criblage de catalyseurs, ii) une étude de type structure-activité sur des séries de catalyseurs, basée sur des caractérisations structurales, texturales et de surface, iii) une étude mécanistique et iv) une étude cinétique. Un protocole de mesure d'activité adapté aux faibles conversions est développé. Le criblage catalytique met en évidence l'activité des solutions solides de cérine-zircone (CZ). Les CZ préparées par pyrolyse de flamme sont un ordre de grandeur plus actives que les CZ préparées par coprécipitation. De façon surprenante, il n'est pas possible a priori de distinguer des catalyseurs très actifs et peu actifs à partir de leurs caractéristiques structurales et texturales. Cependant, on observe que l'activité est corrélée à la densité et à la nature des espèces de surfaces méthoxides et carbonates. Le mécanisme réactionnel de l'état de l'art est affiné grâce à des mesures d'échanges isotopiques et de DRIFT. L'étude cinétique intègre des corrections thermodynamiques de l'équilibre de phases du binaire MeOH-CO2 qui constitue un liquide expansé / Dimethylcarbonate (DMC) is used in polymer synthesis as well as a fuel additive and solvent. The current industrial DMC production is a polluting and hazardous process. On the other hand, the direct carboxylation of methanol with CO2 is a green route to DMC. However, this reaction is highly limited by thermodynamics, limiting the conversion to less than 1%. The integration of a catalyst in a catalytic membrane reactor for water and DMC removal, would shift the equilibrium conversion thereby improving the DMC yield. The aim of this thesis is to develop and study highly active catalysts for DMC synthesis. This manuscript covers: (i) catalyst screening (ii) a structure-activity relationship study (iii) a mechanistic approach and (iv) a kinetic study. A protocol to measure the activity at low conversion has been developed. Catalyst screening evidenced solid solutions of ceria/zirconia (CZ) as the most active and selective. Flame sprayed pyrolysis ceria/zirconia are one order of magnitude more active than coprecipitated CZ. Interestingly, structural and textural features like crystalline and porous structure were similar regardless of the activity. Nevertheless, the activity could be correlated to the nature and the density of the methoxides and carbonates surface species. The mechanism was refined by isotopic exchange and DRIFT experiments. A kinetic study was performed in a batch reactor that integrated the physical equilibria of the gas-expanded reacting mixture

>Magister Scientiae - MSc / New therapeutic strategies are needed for a diverse array of poorly understood neurological impairments. These include neurodegenerative disorders such as Parkinson’s disease and Alzheimer’s disease, and the psychiatric disorders such as depression, anxiety and drug dependence. Popular neuropharmacotherapies have focused on dopamine (DA), serotonin (5HT), γ-aminobutric acid (GABA) and glutamate systems (Jupp & Lawrence, 2010). However recent research points to the sigma receptor (σR) as a possible neuromodulatory system. Due to its multi-receptor action, the σR can trigger several significant biological pathways. This indicates its ideal potential as a drug target to effectively minimise drug dosage and potential side effects. Currently there are a limited number of σR ligands available and few possess the selectivity to significantly show σR’s role in neurological processes. Polycyclic amines have shown notable sigma activity and provide an advantageous scaffold for drug design that can improve pharmacodynamic and pharmacokinetic properties (Banister et al., 2010; Geldenhuys et al., 2005). Aryl-heterocycle amine groups were also shown to improve σR activity (Piergentili et al., 2009).