Humans are exposed to a variety of chemicals including environmental pollutants, cosmetics, food preservatives and drugs. Some of these substances might be harmful to the human body. Traditional toxicological and behavioural investigations performed in animal models are not suitable for the screening of a large number of compounds for potential toxic effects. There is a need for simple and robust in vitro cellular models that allow high-throughput toxicity testing of chemicals, as well as investigation of specific mechanisms of cytotoxicity. The overall aim of the thesis has been to evaluate neuronally differentiated mouse embryonal carcinoma P19 cells (P19 neurons) as a model for such testing. The model has been compared to other cellular models used for neurotoxicity assessment: retinoic acid-differentiated human neuroblastoma SH-SY5Y cells and nerve growth factor-treated rat pheochromocytoma PC12 cells. The chemicals assessed in the studies included the neurotoxicants methylmercury, okadaic acid and acrylamide, the drug of abuse MDMA (“ecstasy”) and a group of piperazine derivatives known as “party pills”. Effects of the chemicals on cell survival, neurite outgrowth and mitochondrial function have been assessed. In Paper I, we describe a fluorescence-based microplate method to detect chemical-induced effects on neurite outgrowth in P19 neurons immunostained against the neuron-specific cytoskeletal protein βIII-tubulin. In Paper II, we show that P19 neurons are more sensitive than differentiated SH-SY5Y and PC12 cells for detection of cytotoxic effects of methylmercury, okadaic acid and acrylamide. Additionally, in P19 neurons and differentiated SH-SY5Y cells, we could demonstrate that toxicity of methylmercury was attenuated by the antioxidant glutathione. In Paper III, we show a time- and temperature-dependent toxicity produced by MDMA in P19 neurons. The mechanisms of MDMA toxicity did not involve inhibition of the serotonin re-uptake transporter or monoamine oxidase, stimulation of 5-HT2A receptors, oxidative stress or loss of mitochondrial membrane potential. In Paper IV, the piperazine derivatives are evaluated for cytotoxicity in P19 neurons and differentiated SH-SY5Y cells. The most toxic compound in both cell models was TFMPP. In P19 neurons, the mechanism of action of TFMPP included loss of mitochondrial membrane potential. In conclusion, P19 neurons are a robust cellular model that may be useful in conjunction with other models for the assessment of chemical-induced neurotoxicity.

Zum gegenwärtigen Kenntnisstand bezüglich Solvatochromie, Sol-Gel Prozesse, und der Synthese von Polyketonen wird eine kurze Einführung gegeben. Die Synthesekonzeptionen funktionalisierter aromatischer Aminoketone wereden vorgestellt. Die neun Verbindungen wurden mittels Elementaranalyse, Röntgenstrukturanalyse, und spektroskopischen (NMR, UV/Vis, MS) Methoden aufgeklärt. Im Mittelpunkt der Untersuchungen steht die Untersuchung des Einflusses von unterschiedlichen Medien (Lösungmittel, Oberflächen, Sol-Gel Materialien und Nachbarnmoleküle im Kristall) auf die Lage der UV/Vis-Absorptionsmaxima verschiedener aromatischer Aminoketone. Die Ergebnisse der Untersuchungen liefern Informationen in Bezug auf das spezifische Solvatationsvermögen, die Polarität von Feststoffoberflächen, der Einfluss funktionaler Gruppen in aromatischen Aminoketonen auf die intermolekulare Wasserstoffbrückenbindungen in Kristallen, und über die Natur der Gast-Host-Wechselwirkungen. Auf der Basis von nucleophilen Substitutionsreaktionen wurden zwei verschiedene Prozesse für die Synthese von Ploy(benzophenone-co-piperazin) und der Kompositform entwickelt. Molekulare Strukturen und Eigenschaften konnten durch Elementaranalyse, mehrere spektroskopische (IR, Festkörper-NMR, UV/Vis, MALDI-TOF) Methoden, Zetapotentialmessungen in wässeriger Phase und thermogravimetrischen Bestimmungen charakterisiert werden.

Tuberculosis affects millions of people worldwide every year. The current treatment for TB is divided into a regimen of both first- and second-line drugs, where first-line treatments are more tolerated and require shorter treatment lengths. With rising levels of resistance, alternative treatment regimes are urgently needed to fight this disease. Ethionamide, a second-line drug is administered as a prodrug which is activated in vivo by the enzyme EthA, which is in turn regulated by EthR. The disruption of the action of EthR could lead to novel therapeutics which could enhance the efficacy of ethionamide, and raise it to a first-line treatment. The work reported in this thesis examines the elaboration of three chemical scaffolds using fragment-based approaches to develop novel inhibitors capable of disrupting the EthR-DNA interaction. The first scaffold, 5-(furan-2-yl)isoxazole was investigated by fragment-merging approaches and produced compounds with the best of these having a KD of 7.4 uM. The second scaffold, an aryl sulfone was elaborated using fragment-merging strategies. This led to several modifications of the fragment, leading to several variants with KDs around 20 uM. With both of these series the affinity could not be improved below 10 uM and due to the synthetic complexity a further scaffold was prioritised. The third scaffold was explored was a 4-(4-(trifluoromethyl)phenyl)piperazine using fragmentgrowing from the NH of the piperazine to probe deeper into the EthR binding pocket. In addition to this, SAR around the 4-(trifluoromethyl)phenyl group was assessed to explore the interactions with EthR. These modifications led to compounds with nanomolar IC50s. A range of compounds were then screened by REMAssay to determine the boosting effect on ethionamide, and this identified compounds with up to 30 times boosting in the ethionamide MIC. The final chapter examines a concept where compounds were designed to exploit the dimeric nature of EthR by linking two chemical warheads with a flexible linker. These compounds are examined using mass spectrometry to investigate the stoichiometry of the interaction to provide insight into the binding of these extended compounds and exploring an alternative strategy to inhibit EthR. The work in this thesis demonstrated the successful use of fragment-based approaches for development of novel EthR inhibitors which showed significant ethionamide boosting effects.

Submitted by JÚLIO HEBER SILVA (julioheber@yahoo.com.br) on 2016-12-07T17:58:06Z No. of bitstreams: 2 Dissertação - Patrícia Caixeta Castro Souza Braga - 2016.pdf: 1832524 bytes, checksum: ad2e9c0895bcec4bc58f82b51c27f966 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2016-12-13T15:39:33Z (GMT) No. of bitstreams: 2 Dissertação - Patrícia Caixeta Castro Souza Braga - 2016.pdf: 1832524 bytes, checksum: ad2e9c0895bcec4bc58f82b51c27f966 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-12-13T15:39:33Z (GMT). No. of bitstreams: 2 Dissertação - Patrícia Caixeta Castro Souza Braga - 2016.pdf: 1832524 bytes, checksum: ad2e9c0895bcec4bc58f82b51c27f966 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-08-26 / Faced with the high and increasing number of people suffering from some form of mental illness in the world, it is necessary to develop additional therapeutic options for patients not helped by existing treatments and also to address medical / famacológicas unmet needs. Given this panorama, this paper proposes the design, synthesis and evaluation of pharmacological type anxiolytic activity and antioxidant profile of a new drug candidate prototype 4- (3,5-ditert-butyl-4-hydroxybenzyl) piperazine-1 carboxylate acetate (LQFM180 (8)). The new prototype was designed by molecular hybridisation strategy of prototypes from 4 - ((1-phenyl- 1H-pyrazol-4-yl) methyl) piperazine-1-carboxylate Ethyl (LQFM008 (5)) and 2,6-di- tertbutyl-phenol (BHT (9)). LQFM180 (8) The compound was synthesized by the Mannich reaction, in 92% yield, which was chemically characterized by infrared spectroscopy (IR) and nuclear magnetic resonance dimensional and two-dimensional (1H, HSQC and HMBC). Evaluation of antioxidant status was carried out by cyclic voltammetry, which confirmed that the compound has antioxidant activity. For evaluation central pharmacological activity of the compound were worked four behavioral models in animals, with treatment with LQFM180 at doses of 25, 50 and 100 mmol / kg V.O. In the test of sleep induced by sodium pentobarbital, the LQFM180 (8) reduced latency and increased barbiturate sleep duration, indicating a central depressant activity. Treatment with LQFM180 (8) in different doses did not alter the number of self-cleaning, dung, total and raised intersections, behavioral parameters observed in the open field test; there is no impairment of exploratory activity. Also in the open field test, the compound LQFM180 (8) indicated anxiolytic type activity, demonstrated by the increase in length of stay, and the entrance in the center of the field. LQFM180 (8) treatment did not alter the motor activity test in the chimney, which was evidenced by the animal climbing time. The compound LQFM180 (8) evaluated the maze test in high cross, possess anxiolytic activity type; evidenced by the increase in time and entering the open arms and the time reduction in the central platform. At the end of this work we can see that the synthetic route proposed for obtaining LQFM180 (8) compound was effective, given the high yields obtained, little laborious steps and cost. Finally, we conclude that the employee structural planning was ratified before the success of the structural characterization of the compound and the results obtained from the central pharmacological evaluation in animal models. As perspective, it is necessary to establish the mechanism of action of the molecule as well as the continuation of in vivo evaluations. / Diante do elevado e crescente número de pessoas que sofrem com algum tipo de doença mental no mundo, faz-se necessário o desenvolvimento de opções terapêuticas adicionais para pacientes não ajudados por tratamentos já existentes e também para abordar necessidades médico/famacológicas não satisfeitas. Diante deste panorama, este trabalho propõe o planejamento, síntese e avaliação das atividades farmacológicas tipo ansiolíticas e do perfil antioxidante de um novo candidato a protótipo de fármaco 4-(3,5-di-terc-butil-4- hidroxibenzil)piperazina-1-carboxilato de etila (LQFM180 (8)). O novo protótipo foi planejado através da estratégia de hibridação molecular a partir dos protótipos 4-((1-fenil-1Hpirazol-4-il)metil)piperazina-1 carboxilato de etila (LQFM008 (5)) e do 2,6-di-tert-butil-fenol (BHT (9)). O composto LQFM180 (8) foi sintetizada através da reação de Mannich, em 92% de rendimento, o qual foi caracterizado quimicamente por espectroscopia no infravermelho (IV) e ressonância magnética nuclear unidimensional e bidimensional (1H, HMBC e HSQC). A avaliação do perfil antioxidante foi realizada através de voltametria cíclica, a qual confirmou que o composto apresenta atividade antioxidante. Para avaliação de atividade farmacológica central do composto foram trabalhados quatro modelos comportamentais em animais, com tratamento com LQFM180 (8) nas doses de 25, 50 e 100 µmol/kg v.o. No teste do sono induzido por pentobarbital sódico, o LQFM 180 (8) reduziu o tempo de latência e aumentou o tempo de duração do sono barbitúrico, indicando uma atividade depressora central. O tratamento com LQFM180 (8), nas diferentes doses, não alterou o número de autolimpeza, bolos fecais, cruzamentos totais e levantadas, parâmetros comportamentais observados no teste do campo aberto; não havendo comprometimento da atividade exploratória. Também no teste do campo aberto, o composto LQFM180 (8) indicou atividade tipo ansiolítica, demonstrado pelo aumento no tempo de permanência, e na entrada no centro do campo. O tratamento com LQFM180 (8) não alterou a atividade motora do animal quando avaliado no teste da chaminé, o que foi evidenciado pelo não alteração no tempo de escalada do animal. O composto LQFM180 (8), avaliado pelo teste de labirinto em cruz elevado, possui atividade tipo ansiolítica; evidenciado pelo aumento no tempo e na entrada nos braços abertos, e pela redução do tempo na plataforma central. Ao término deste trabalho podemos observar que a rota sintética proposta para a obtenção do composto LQFM180 (8) se mostrou eficaz, face ao alto rendimento obtido, etapas pouco laboriosas e de baixo custo. Por fim, podemos concluir que o planejamento estrutural empregado foi ratificado, diante do êxito na caracterização estrutural do composto e dos resultados obtidos da avaliação farmacológica central realizada em modelos animais. Como perspectivas, há que se estabelecer o mecanismo de ação da molécula, bem como a continuação das avaliações in vivo.