Deregulated activation of phosphatidylinositol 3-kinase (PI3K) pathway is central to many human malignancies. The functions of this pathway are critical for normal cell metabolism, proliferation, and survival. In lung cancers, the PI3K pathway activity is often aberrantly driven by multiple mutations, including EGFR, KRAS, and PIK3CA. Molecules targeting the PI3K pathway are intensely investigated as potential anti-cancer agents. Although inhibitors of the pathway are currently in clinical trials, rational and targeted use of these compounds, alone or in combination, requires an understanding of isoform-specific activity in context. We sought to identify class IA PI3K enzyme (p110a/PIK3CA, p110b/PIK3CB, p110d/PIK3CD) activities using isoform-specific inhibitors in a lung cancer model system. Treatment of non-small cell lung cancer (NSCLC) cell lines with PIK3CA, PIK3CB, PIK3CD or PIK3CB/D inhibitors resulted in pharmacokinetic and pharmacodynamic responses that frequently tracked with a specific mutation status. Activation of PIK3CA dictated response to the PIK3CA-specific inhibitor while deletion of PTEN phosphatase indicated response to the PIK3CB inhibitor. The PIK3CD isoform-specific inhibitors lacked efficacy in all NSCLC cell lines tested, however treatment at increased concentrations likely provide concurrent inhibition of both PIK3CB/D isoforms improving activity of either agent alone but did not track with a single biomarker. The observed pharmacodynamic and proliferation responses to isoform-specific inhibitors suggested that PI3K isoforms may functionally compensate for loss of another in certain genetic backgrounds. These studies demonstrate unanticipated cellular responses to PI3K isoform inhibition in NSCLC, suggesting that patient populations with specific mutations can benefit from certain isoform-selective inhibitors, or combinations, allowing for rational and targeted clinical use of these agents.

Natural products provide some of the most potent anticancer agents and offer a template for new drug design or improvement with the advantage of an enormous chemical space. The overall goal of this thesis research is to enhance the chemical space of two natural products in order to generate novel drugs with better in vivo bioactivities than the original natural products. Polycarcin V (PV) is a gilvocarcin-type antitumor agent with similar structure and comparable bioactivity with the principle compound of this group, gilvocarcin V (GV). Modest modifications of the polyketide-derived tetracyclic core of GV had been accomplished, but the most challenging part was to modify the sugar moiety. In order to solve this problem, PV was used as an alternative lead-structure for modification because its sugar moiety offered the possibility of enzymatic O-methylation. We produced four PV derivatives with different methylation patterns for cytotoxicity assays and provided important structure-activity-relationship information. Mithramycin (MTM) is the most prominent member of the aureolic acid type anticancer agents. Previous work in our laboratory generated three MTM analogues, MTM SA, MTM SK, and MTM SDK by inactivating the mtmW gene. We developed new MTM analogues by coupling many natural and unnatural amino acids to the C-3 side chain of MTM SA via chemical semi-synthesis and successfully made some compounds with both improved bioactivity and in vivo tolerance than MTM. Some of them were consequently identified as promising lead-structures against Ewing’s sarcoma. The potential of selectively generating novel MTM analogues led us to focus on a key enzyme in the biosynthetic pathway of mithramycin, MtmC. This protein is a bifunctional enzyme involved in the biosynthesis of TDP-D-olivose and TDP-D-mycarose. We clarified its enzymatic mechanisms by X-ray diffraction of several crystal complexes of MtmC with its biologically relevant ligands. Two more important post-PKS tailoring enzymes involved in the biosynthesis of the MTM side chains, MtmW and MtmGIV, are currently under investigation. This would not only give us insight into this biosynthetic pathway but also pave the way to develop potentially useful MTM analogues by engineered enzymes.

Chapter 1 gives background information on proteases and discusses the concept of protease inhibition as a therapeutic strategy for humans. It introduces the key concept that conformation defines biological activity. It also outlines how proteases almost universally bind their substrate/inhibitors in an extended β-strand conformation. The use of calpain as a prototype protease for the testing of β-strand mimics synthesised later in the thesis is also discussed. Chapter 2 describes how molecular modeling was used to rationalise the structure based activity relationships (SAR) of known calpain inhibitors. Molecular modeling was then used to successfully design a number of acyclic β-strand mimics. The synthesis and testing of eight such inhibitors is described. The most potent β-strand mimic prepared was 2.13. This was determined to have an IC₅₀ of 30 nM against calpain II. Chapter 3 outlines the history and application of ring closing metathesis (RCM) to the synthesis of cyclic compounds. The attempted synthesis of an eight membered cyclic nitrogen to nitrogen conformationally constrained dipeptide is described. The synthesis of a conformationally constrained β-amino acid calpain inhibitor (3.73) is also described. A novel calpain inhibitor motif was designed in Chapter 4. On the basis of this an in-silico combinatorial library of two hundred and eighty eight possible β-strand templates was prepared. Conformational analysis of this library was performed and from this a number of excellent β-strand templates were identified and selected for synthesis. The preparation of ten β-strand templates is described. New microwave irradiation methodology was developed to achieve this. vii The formation of a six-membered catalyst deactivating chelate is also proposed to explain why some dienes fail to undergo RCM. Two methods to circumvent the formation of such a chelate are outlined. The addition of Lewis acid chloro-dicyclohexyl borane to the RCM reaction mixture and chain length alteration are investigated. Chapter 5 describes the design of macrocyclic β-strand mimics using induced fit molecular modelling. The physicochemical properties of these were calculated in-silico. From this analysis a number of Tyr-XX-Gly based and Tyr-XX-Cys based macrocyclic calpain inhibitors were selected for synthesis. The preparation and testing of these are described. In the Tyr-XX-Gly macrocyclic system a number of variables were investigated and numerous SAR implications concluded. Aldehyde 5.14 was identified as the best electrophilic warhead macrocyclic calpain inhibitor with an IC₅₀ against calpain II of 27 nM. The best non-electrophilic warhead macrocycle (5.13) had an IC₅₀ against calpain II of 704 nM. Chapter 6 describes synthetic optimisation for the preparation of calpain inhibitors 2.13, 5.14 and 5.17. Multi-gram quantities of each were prepared. Aldehydes 2.13 and 5.14 were evaluated as anti-cataract agents using in-vivo cataract sheep model. Both of these β-strand mimics were demonstrated to retard cataract development. Macrocycle 5.14 was found to be the most effective, decreasing the rate of cataract development between forty four and forty nine per cent relative to control. Chapter 7 outlines the attempted development of RCM methodology for the chiral synthesis of α-α disubstituted amino acid lactams. In addition, methodology for the stereoselective incorporation of a C-N constrained β-amino acid carbocycle into a peptide or peptidomimetic is described.

Careful treatment of water molecules in ligand-protein interactions is required in many cases if the correct binding pose is to be identified for molecular docking. Water can form complex bridging networks and can play a critical role in dictating the binding mode of ligands. A particularly striking example of this can be found in the ionotropic glutamate receptors (iGluRs), a family of ligand gated ion channels that are responsible for a majority of the fast synaptic neurotransmission in the central nervous system that are thought to be essential in memory and learning. Thus, pharmacological intervention at these neuronal receptors is a valuable therapeutic strategy. This thesis relies on various computational studies and X-ray crystallography to investigate the role of ligand-water mediated interactions in iGluRs bound to glutamate and α-amino-3-hydroxy-5-methyl-4- isoxazole-propionic acid (AMPA). Comparative molecular dynamics (MD) simulations of each subtype of iGluRs bound to glutamate revealed that crystal water positions were reproduced and that all but one water molecule, W5, in the binding site can be rearranged or replaced with water molecules from the bulk. Further density functional theory calculations (DFT) have been used to confirm the MD results and characterize the energetics of W5 and another water molecule implicated in influencing the dynamics of a proposed switch in these receptors. Additional comparative studies on the AMPA subtypes of iGluRs show that each step of the calculation must be considered carefully if the results are to be meaningful. Crystal structures of two ligands, glutamate and AMPA revealed two distinct modes of binding when bound to an AMPA subtype of iGluRs, GluA2. The difference is related to the position of water molecules within the binding pocket. DFT calculations investigated the interaction energies and polarisation effects resulting in a prediction of the correct binding mode for glutamate. For AMPA alternative modes of binding have similar interaction energies as a result of a higher internal energy than glutamate. A combined MD and X-ray crystallographic study investigated the binding of the ligand AMPA in the AMPA receptor subtypes. Analysis of the binding pocket show that AMPA is not preserved in the crystal bound mode and can instead adopt an alternative mode of binding. This involves a displacement of a key water molecule followed by AMPA adopting the pose seen by glutamate. Thus, this thesis makes use of various studies to assess the energetics and dynamics of water molecules in iGluRs. The resulting data provides additional information on the importance of water molecules in mediating ligand interactions as well as identifying key water molecules that can be useful in the de novo design of new selective drugs against iGluRs.

This work seeks to contribute to the discipline of neuropharmacology by way of structure activity relationship from the standpoint of an organic chemist. More specifically, we sought to develop robust synthetic methodology able to efficiently produce an array of compounds for the purpose of systematic evaluation of their interaction with specific sights within the central nervous system (CNS) in order to better understand the mind and to develop drugs that may have beneficial effects on neurological function. The focus of these studies has been toward the development of novel molecules, using a structure-activity relationship approach, that exhibit binding affinity at specific targets within the CNS. The merit of such studies is twofold: primarily, new compounds are produced that provide valuable scientific insight about their physiological targets, and secondarily, new synthetic methodologies that may arise in order to produce these compounds, thereby contributing to the whole of organic chemistry. As a result of the research described herein, the development of one high affinity and several moderate affinity compounds at the cannabinoid receptor subtype 1 (CB1) has been accomplished. The research demonstrates that a diaryl ether molecular scaffold represents a successful motif in the cannabinoid pharmacophore. The production of the compounds in the SAR studies also introduced a novel general synthetic methodology for the synthesis of diaryl ethers around a phloroglucinol core. A second project was initiated in order to explore the synthetic methods required to develop a general process for the synthesis of rigid aminobenzocyclobutane analogs of known phenethylamines with activity at monoaminergic neurotransmitter sites. Using the synthetic approach devised here, four novel aminobenzocyclobutane isomeric analogs of a known pharmacologically active phenethylamine, (RS)-phenylpropan-amine were synthesized and are currently being evaluated for pharmacological potential.

Au cours de ce travail, nous avons développé des voies de synthèses convergentes et énantiosélectives en vue de préparer des analogues pyrrolidiniques des alcaloïdes de Lobelia comme nouveaux ligands des récepteurs nicotiniques à l’acétylcholine. Deux familles de ligands ont été réalisées par des méthodes d’élongation mono- ou bi-directionnelle basées respectivement sur des stratégies de désymétrisation précoce ou tardive au départ du succinaldéhyde.La première partie de ce manuscrit aborde la conception d’hybrides de lobéline, nicotine et d’agonistes naturels. Ces structures originales ont été obtenues diastéréosélectivement grâce à un intermédiaire commun issu d’une élongation monodirectionnelle du succinaldéhyde. Cette voie exploitera la chimie des iminiums masqués. La mise au point de cette synthèse s’est enrichie par la découverte et la valorisation d’une nouvelle famille de ligands chimériques.La seconde partie étudie la voie d’élongation bidirectionnelle basée sur des réactions de double aza-Michael suivies de la réduction désymétrisante tardive de pyrrolidines 2,5-phénacyl méso et pseudo-méso. Cette stratégie asymétrique s’inscrit dans une démarche d’économie d’atomes et d’étapes. La perspective majeure de ce travail est l’évaluation par électrophysiologie sur différents sous-types de récepteurs à l’acétylcholine d’une sélection de ligands hybrides.Les études de RSA menées sur ces familles de composés de haute homologie structurale permettront in fine d’améliorer les modèles prédictifs décrivant les transitions allostériques des récepteurs nicotiniques à l’acétylcholine / During this PhD work, convergent and diastereoselective routes for the preparation of pyrrolidine Lobelia alkaloid analogues have been developed as novel neuronal nicotinic receptor ligands. Two families of ligands have been synthesized by a strategy of mono- or bi-directional elongation of the succinaldehyde including early or late desymmetrization process respectively.The first part of this manuscript is dedicated to the preparation of hybrids of lobeline, nicotine and other natural agonists. These original structures have been diastereoselectively obtained thanks to a common intermediate resulting of the mono-elongation of succinaldehyde. This synthetic pathway uses the chemistry of masked iminium. The development of this strategy has been enriched by the discovery and the valorisation of a new chimeric ligand family.The second part studies the “bidirectional” elongation route, based on a ring-closing double aza-Michael reaction followed by the desymmetrizing reduction of meso and pseudo-meso 2,5-diphenacyl pyrrolidines. This asymmetric strategy constitutes a step- and atom-economical approach. The major perspective of this work is the biological evaluation of selected ligands by electrophysiology made on different nAChR subtypes.The SAR studies realized on these structurally homologue ligand families could allow the improvement of the predictive molecular models describing the allosteric conformations of the nAChRs.

A Doença de Chagas, causada pelo parasito tripanossomatídeo Trypanosoma cruzi, é endêmica e se distribuí por toda América Latina. É uma das parasitoses mais negligenciadas pela indústria farmacêutica e os únicos fármacos disponíveis para seu tratamento foram introduzidos há décadas. Infelizmente, eles são ineficientes e apresentam sérios efeitos colaterais. Esse panorama mostra a necessidade do desenvolvimento de novos fármacos para a quimioterapia contra a doença de Chagas. As enzimas pertencentes a vias metabólicas essenciais para a sobrevivência do parasito tais como a via glicolítica e a de síntese de novo de nucleotídeos de pirimidinas, têm sido propostas como alvos interessantes no planejamento novos fármacos para o tratamento da doença de Chagas. Neste trabalho, as enzimas Gliceraldeído 3-fosfato desidrogenase (TcGAPDH) e a Diidroorotato desidrogenase (TcDHODH) de Trypanosoma cruzi foram estudadas como alvos para o planejamento de inibidores enzimáticos com propriedades físico-químicas e características estruturais similares à de compostos-líderes. Para isso, foram utilizados métodos e ferramentas de Quiminformática tanto baseadas nas estruturas dos ligantes (LBVS) quanto dos receptores (SBVS). Para a identificação e seleção de potenciais inibidores da enzima GAPDH, uma coleção virtual obtida do banco de dados ZINC, contendo aproximadamente 2,5 milhões de compostos, foi avaliada através de vários filtros de seleção com o objetivo de priorizar aqueles compostos mais interessantes do ponto de vista estrutural, de propriedades físico-químicas e farmacocinéticas. A aplicação desses filtros originou uma subcoleção de aproximadamente 450 mil estruturas que foram avaliadas segundo a complementaridade de interações com a estrutura da enzima através de métodos de docagem molecular. Com base nestes resultados, doze compostos que se mostraram promissores foram selecionados e adquiridos comercialmente para serem testadas in vitro contra a enzima TcGAPDH. Dos doze compostos testados, três exibiram afinidade (Ki) pela enzima em concentrações inferiores a 80 μM, Além disso, esses compostos também são caracterizados pelo baixo peso molecular (274 a 330 g mol-1) e no máximo 24 átomos diferentes do hidrogênio e, como consequência, apresentam eficiências do ligante entre 0,24 e 0,34 Kcal mol-1 átomo-1, o que os tornam ótimos candidatos à otimização molecular visando aumento da afinidade pelo alvo. Para a busca de inibidores da enzima TcDHODH, primeiramente foi realizada uma busca por cavidades na estrutura 3D do alvo para a identificação de regiões distintas do sítio catalítico e passíveis de serem exploradas no planejamento de ligantes. Através desta análise foi possível o estabelecimento de quatro novas regiões com forma, volume e localizações adequadas para acomodar pequenas moléculas capazes de modular a atividade da TcDHODH. Uma destas regiões, chamada S2, localizada sob a alça β4-αA e no canal de acesso dos substratos ao sítio ativo, foi escolhida para o planejamento baseado na estrutura do alvo. As estruturas de aproximadamente cem compostos derivados de pirimidinas, substituídos em posições estrategicamente definidas e selecionados através de buscas por subestruturas, foram docadas no sítio de interesse e nove compostos adquiridos e testados in vitro contra a enzima com o objetivo de validar as hipóteses estabelecidas inicialmente. Destes, cinco compostos mostraram potências (IC50) superiores à do produto de reação (inferior a 150 μM), Os resultados encontrados validaram as hipóteses geradas na primeira etapa e foram usados para direcionar a seleção de outras quinze novas moléculas através de um protocolo de docagem molecular com ajuste induzido. A avaliação in vitro desses compostos contra a enzima TcDHODH resultou na identificação de outros 11 compostos ativos, dos quais o mais potente exibiu afinidade pela enzima em concentração igual a 124 nM. Este composto possui eficiência do ligante igual a 0,56 Kcal mol-1 átomo-1 e pode ser considerado um fragmento molecular com excelentes características do ponto de vista do potencial para futuro desenvolvimento como agente terapêutico. Os resultados encontrados também evidenciaram a importância de determinadas características estruturais impressas nos inibidores da TcDHODH para a complementaridade com o novo sítio de interação identificado. Assim, novos compostos foram propostos para otimização molecular com o objetivo de melhorar afinidade e aumentar a diversidade de classes e, deste modo, ampliar o espectro de perfis farmacocinéticos para posteriores ensaios celulares e in vivo, Através da realização deste trabalho foi possível validar as estratégias adotadas na utilização dos métodos computacionais e também as hipóteses construídas a partir da aplicação dos mesmos. A taxa de acerto (TA) alcançada foi superior a 30% no planejamento de inibidores para ambos os alvos, ou seja, muito superiores às encontradas em experimentos de ensaio em massa. Deste modo, este estudo contribuiu com a proposição de novos esqueletos moleculares que podem ser usados como compostos-líderes no desenvolvimento de novos agentes tripanocidas focando nas enzimas TcGAPDH e TcDHODH como alvos. / Chagas\' disease, an endemic illness widely distributed throughout Latin America, is caused by the protozoa parasite Trypanosoma cruzi. It is one of the tropical diseases that are among the most neglected by the pharmaceutical industry, for which available treatments were launched more than 30 years ago. In addition, these drugs are ineffective and cause severe side effects to patients. This panorama shows the need for the development of new and more effective chemotherapeutic agents for the treatment of the disease. Enzymes belonging to metabolic pathways that are essential for the parasite survival such as the glycolysis and pyrimidine nucleotide biosynthesis have been proposed as attractive targets for the design of new drugs for the treatment of Chagas disease. In this work, the enzymes Gyceraldehyde-3-phosphate dehydrogenase (TcGAPDH) and the Dihydroorotate dehydrogenase (TcDHODH) from Trypanosoma cruzi were studied as targets for the design of inhibitors with physicochemical properties and structural characteristics similar to lead-compounds. Methods in Cheminformatics within the Ligand- and Structure-based Virtual Screening (LBVS and SBVS, respectively) approaches were thoroughly employed as tools to identify new hits. For the selection and identification of potential inhibitors of the GAPDH enzyme, a compound database containing nearly 2.5 million of small molecules retrievable from ZINC was evaluated through several molecular filters aiming at prioritizing those compounds more interesting from the point of view of their structures, physicochemical and predicted ADME/Tox properties. The application of Filter originated a subcollection of approximately 450 thousand structures that were then scored according to their complementary interactions with the 3D structure of the enzyme through molecular docking. Based on docking results, twelve compounds that showed to be promising ligands were selected and commercially acquired for in vitro assays against the TcGAPDH. Of the twelve compounds evaluated in vitro, three exhibited affinity constants (Ki) at concentrations lower than 80 μM. Furthermore, the selected compounds are also characterized by the low molecular weight (274 to 330 g mol-1) and a maximum of non-hydrogen atom count of 24, as a result, they have Ligand Efficiencies between 0,24 and 0,34 Kcal mol-1 átomo-1, what grant them great potential as candidates for molecular optimization and potency improvement. For the search of TcDHODH inhibitors, cavities in the 3D structure of the target for the identification of areas apart from catalytic site but likely to be explored in the design of ligands were selected a priori. This resulted in four new regions with appropriate shape, volume and locations to accommodate small molecules capable of modulating the activity of TcDHODH. One of the areas, called S2 site, is located under the α4 - βA loop and in the access channel of the substrate to the active site and was chosen to be explored in the SBDD studies. Approximately one hundred of pyrimidine derivatives containing strategically defined posítions for molecular substitution were retrieved from commercially available compounds database through substructure searching and docked into the previously defined site. Based on the docking results nine compounds were selected, purchased and assayed in vitro against the enzyme with the objective of validating the hypothesis. Of these, five compounds showed potencies (IC50) better than that exhibited by the product of the reaction (values lower than 150 μM). Thus, the results found validated the hypotheses generated in the first stage of the designing and they were used to drive the selection of other fifteen new molecules through induced fit molecular docking protocol. The in vitro evaluation of those compounds against the TcDHODH enzyme resulted in the identification of other eleven ligands, of which the most potent exhibited affinity for enzyme at the concentration of 124 nM. This molecule has a Ligand Efficiency of 0.56 Kcal mol-1 atom-1 and can be considered a fragment-like compound with excellent characteristics from the point of view of its potential for future development as therapeutic agent. The results found also evidenced the importance of certain structural characteristics in the inhibitor of TcDHODH for the complementarily with the new identified site of interaction. Thus, new compounds were proposed for potency and class diversity improvement. By doing so we hope to enlarge ADME profile spectrum for further cellular and in vivo assays. Through the success of this work, it was possible to validate the strategies adopted in the use of computational methods and also the hypotheses built from the application of that. The success rate (TA) obtained was higher than 30% in the design of ligands for both studied targets, which is much better than that usually found along High Throughput Screening assays. Thus, this study contributed with the proposítion of new molecular scaffolds that can be used as lead compounds in the development of new tripanocidal agents having as targets the enzymes TcGAPDH or TcDHODH.

A esquistossomose é uma das principais causas de morbidade em países Tropicais e Subtropicais, gerando graves consequências socioeconômicas. Atualmente, os fármacos disponíveis para o tratamento da desta doença são praziquantel e oxamniquina, porém relatos de baixa susceptibilidade do parasita a esses medicamentos sugerem a necessidade de novas estratégias terapêuticas para o tratamento da doença. Todavia, existe pouco interesse da indústria farmacêutica no desenvolvimento de fármacos contra doenças tropicais e negligenciadas, entre as quais se encontra a esquistossomose. Devido a estes fatores, o presente trabalho teve por objetivo geral utilizar ferramentas computacionais para identificar inibidores da SmDHFR candidatos a novos fármacos. Avaliou-se as características exclusivas para a proteína de S. mansoni por meio de uma análise das sequências FASTA em comparação com a DHFR de outros organismos. A fim de garantir a ação seletiva dessas moléculas frente a enzima do parasita, os campos moleculares de interação seletivos para SmDHFR foram calculados e empregados na construção do modelo farmacofórico, o qual foi utilizado na triagem virtual de inibidores de SmDHFR. Os estudos computacionais realizados nos permitiram a seleção de 20 moléculas com uma boa complementariedade com o modelo farmacofórico gerado e com potencial para serem inibidores de SmDHFR. / Schistosomiasis is one of morbidity\'s main causes in tropical and subtropical countries, which leads to serious socioeconomic consequences. Praziquantel and oxamniquina are the drugs currently available for treating this disease, but reports points that the parasite has been resistant to both drugs, which suggests the need for new therapeutic strategies for the treatment of this disease. However, there is little interest in the pharmaceutical industry in developing drugs against neglected tropical diseases, including schistosomiasis. Due to these factors, the present work has the general objective to use computational tools to identify SmDHFR inhibitors which could be good candidates for developing new drugs. Evaluation of the exclusive characteristics of the S. mansoni protein were performed by FASTA sequence analyses in comparison to DHFR from other organisms. In order to guarantee the selective action of these molecules against the parasite enzyme, the molecular interaction fields selective for SmDHFR were calculated and used in the construction of the pharmacophoric model, which was further used in the virtual screening of SmDHFR inhibitors. Computational studies were performed and those led us to 20 molecules with a good complementarity with the pharmacophoric model that was previously generated and with potential to be SmDHFR inhibitors.

Doenças como malária, esquistossomose, filaríase linfática, dengue, leishmaniose e mal de Chagas são endêmicas em países tropicais. Referente ao período entre 1975 e 2004, apenas 1% dos 1.556 novos fármacos registrados foi destinado ao tratamento destas doenças. Somente a doença de Chagas possui aproximadamente 18 milhões de casos em todo mundo. Até o momento, a quimioterapia para o tratamento dessa antropozoonose constitui-se basicamente de dois fármacos, o nifurtimox, proscrito no Brasil, e o benznidazol. Estes fármacos, além de apresentarem vários efeitos colaterais, são pouco eficazes, o que dificulta a adesão dos pacientes ao tratamento. Diante da necessidade imediata de novos fármacos para o combate de tripanossomíase americana, estratégias de modificação molecular têm se mostrado como ferramenta promissora para obtenção de novos fármacos, geralmente inspirados na estrutura molecular de fármacos já conhecidos. A estratégia de modificação molecular permite aumentar as chances de sucesso no processo de descoberta de novos fármacos, reduzindo tempo e custo da pesquisa. Este trabalho teve como objetivo o planejamento, a síntese e a avaliação da atividade anti-T. cruzi e a identificação das propriedades físico-químicas que são responsáveis por modular a atividade anti-T. cruzi da série dos 2-[5-nitrotiofeno- 2-il]-3-acetil-5-[4-fenil-substituído]-2,3-diidro-1,3,4-oxadiazolinas, compostos com estrutura análoga à nifuroxazida, fármaco antimicrobiano que também apresenta atividade antiparasitária. O planejamento da série de compostos estudados foi realizado com base no diagrama de Craig. A obtenção dos derivados 1,3,4-oxadiazolínicos foi realizada em quatro etapas sintéticas: esterificação, amonólise, obtenção da base de Schiff e ciclização oxidativa. As estruturas planejadas foram confirmadas por RMN 1H e RMN 13C, enquanto a pureza foi avaliada pela faixa de fusão e de análise elementar de CHN. A atividade antiparasitária dos compostos foi determinada frente à cepa Y da forma epimastigota do T. cruzi e a concentração de parasitas foi quantificada através da absorbância em espectrofotômetro UV/Vis (λ = 580 nm). Os quinze compostos da série foram avaliados em 6 diferentes concentrações (3,75; 5; 7,5 ; 10; 15; 20 µM), sendo que somente o etoxi-derivado não foi mais ativo que o fármaco padrão, o benznidazol. Entre os compostos analisados identificaram-se o mais e o menos ativo da série, respectivamente: o 2-[5-nitro-tiofeno-2-il]-3-acetil-5-[4 fenil-acetoxi]-2,3-diidro-1,3,4-oxadiazolinas (IC50=7,91 µM) e o 2-[5-nitrotiofeno-2-il]-3-acetil-5-[4 fenil-etoxi]-2,3-diidro-1,3,4-oxadiazolinas ((IC50=26,60 µM). Através de estudos de QSAR-2D, a atividade anti-T. cruzi foi correlacionada com descritores físico-químicos, como a hidrofobicidade, os efeitos eletrônicos e o volume molecular. Com a aplicação de modelos matemáticos, ficou evidenciado que a atividade anti-T. cruzi desta série de compostos sofre notável influência da hidrofobicidade e dos efeitos eletrônico de ressonância, o que permite a continuidade deste trabalho através da variação planejada destas propriedades, visando identificar o análogo mais potente da série de compostos estudados. / Diseases like malaria, schistosomiasis, lymphatic filariasis, dengue, leishmaniasis and Chagas\'disease are endemic in tropical countries. From 1975 to 2004, only 1% of 1.556 new drugs were registered for the treatment of these diseases. Chagas disease itself has approximately 18 million of cases worldwide, and the chemotherapy for the treatment of this anthropozoonosis consists basically of two drugs, nifurtimox, which is prohibited in Brazil, and benznidazole. These drugs have various side effects and can also be ineffective, making patients give up the treatment. Given the immediate need for new drugs to treat american trypanosomiasis, molecular modification strategies have been shown as a promising tool for obtaining new drugs, often inspired by the molecular structure of known drugs. The strategy of molecular modification will increase the chances of success in the discovery process of new drugs, reducing the time and the costs of research. Given the situation above described, this work aims to design synthesis and evaluate the anti-Trypanosoma cruzi activity of new drugs as well as identification physicochemical properties that are responsible for modulating the anti-T. cruzi activity. Series of 2-[5-nitro-thiofen-2-yl]-3-acetyl-5-[4-phenyl-substituted]-2,3-dihydro-1,3,4-oxadiazolines, compounds with similar structure to nifuroxazide, an antimicrobial drug that also has antiparasitic activity. The planning of the series of compounds was carried out based on the Craig diagram. The obtainment of the1,3,4-oxadiazolinics derivatives was performed with four synthetic steps, as follows: esterification, ammonolysis, obtention of the Schiff base and oxidative cyclization. The designed structures were confirmed by 1H and 13C NMR and purity was assessed by melting point and elemental analysis of CHN. The antiparasitic activity of compounds was determined against the Y strain of T. cruzi in the epimastigote form and the concentration of parasites was quantified by absorbance in a spectrophotometer UV / Vis (λ = 580 nm). The fifteen compounds obtained were evaluated in six different concentrations (3.75, 5, 7.5, 10, 15, 20 µM), and only the ethoxy-derivative was less active than the standard drug, benznidazole. The 2-[5-nitrothiofen- 2-yl]-3-acetyl-5-[4-acetoxyphenyl]-2,3-dihydro-1,3,4-oxadiazolines (IC50=7.91µM) and of 2-[5-nitro-thiofen-2-yl]-3-acetyl-5-[4-etoxyphenyl]-2,3-dihydro-1,3,4-oxadiazolines (IC50=26.60 µM) derivatives were identified as the most and the least active of the series, respectively. Through 2D-QSAR studies, the anti-T. cruzi activity was correlated with physicochemical descriptors such as hydrophobicity, molecular volume and electronic effects. Applying mathematical models, it became evident that the activity of anti-T. cruzi activity in this series of compounds undergoes remarkable influence of hydrophobicity and electronic effects of resonance, which allows the continuity of the work planned by varying these properties to identify the most potent analogue among the series of compounds studied.

Este trabalho teve como objetivo a realização de estudos de Relações Quantitativas Tridimensionais Estrutura-Atividade, QSAR 3D, com identificação de alvos potenciais para compostos 5-nitro-heterocíclicos com estruturas azometínica ou oxadiazolínica com bioatividade dual, antifúngica e anti-T. cruzi, visando à identificação de novos compostos que possam ser aproveitados como possíveis candidatos a fármaco ou como compostos-líderes para novos estudos de modificação molecular. Os compostos estudados pertencem a quatro séries intimamente relacionadas, a saber: Série AzoO: 5-nitro-2-furfurilideno benzidrazidas 4-substituídas; Série AzoS: 5-nitro-2-tiofilideno benzidrazidas 4-substituídas; Série OxaO: 3-acetil-oxadiazolina 2,5-furfurilideno benzidrazidas 4-substituídas e Série OxaS: 3-acetil-oxadiazolina 2,5-tiofilideno benzidrazidas 4-substituídas. A utilização de forma integrada de ferramentas pertencentes às áreas de química farmacêutica, quimioinformática e bioinformática permite a caracterização de cavidades catalíticas de proteínas e a identificação das propriedades físico-químicas consideradas essenciais para a interação adequada entre ligante e biomacromolécula-alvo. Por outro lado, permitem também a identificação de alvos a partir da comparação de estruturas químicas, com as propriedades físico-químicas associadas, entre ligantes conhecidos e possíveis alvos; este procedimento de comparação de perfis moleculares, conhecido como virtual profiling, parte do princípio de que moléculas semelhantes a ligantes conhecidos de proteínas têm o potencial de interagir com essa mesma proteína, onde o grau de semelhança é determinado não somente pela estrutura química, mas também pela distribuição de características físico-químicas. As ferramentas utilizadas em estudos in silico incluem também análise de descritores topológicos que permitem a caracterização de propriedades físico-químicas considerando sua distribuição espacial em relação à estrutura química de uma dada molécula. O uso destes descritores permite a determinação do grau de semelhança entre moléculas ou partes de moléculas (fragmentos moleculares), e encontra-se na base das metodologias de triagem e de caracterização de sítios catalíticos de proteínas. Dentro da proposta de trabalho foram realizados estudos envolvendo virtual profiling, análise de fragmentos moleculares com base em descritores físico-químicos capazes de caracterizar superfícies de proteínas e ligantes, caracterização de cavidade catalítica de possível alvo e docking dos compostos a alvos putativos. Foi também realizada a determinação de atividade antifúngica e análise de resultados de QSAR 3D, levando finalmente à construção de uma hipótese quanto ao possível alvo biológico para os compostos azometínicos e oxadiazolínicos. O procedimento de virtual profiling apontou como possíveis alvos as enzimas CYP19A (aromatase), CYP3A4, CYP3A5 e CYP3A7. Embora estas enzimas não estejam diretamente envolvidas com as atividades biológicas testadas para os compostos até o momento, existem estudos que demonstram a interação de compostos azólicos de ação antifúngica, cujo alvo é CYP51 (14α-desmetilase), também com CYP19A e CYP3A4. Em conjunto com a análise da caracterização das propriedades físico-químicas com uso de descritores topológicos e a própria dualidade da atividade biológica, concluiu-se que seria possível a interação dos compostos das séries estudadas com CYP51, sendo esta enzima alvo não somente de antifúngicos azólicos, como também de compostos com ação anti-T. cruzi. A caracterização da cavidade catalítica de CYP51, tanto em termos de descritores de propriedades físico-químicas como de características estereoquímicas, associada ao estudos de QSAR 3D, confirmaram a possibilidade de interação da série oxadiazolínica com a CYP51, em orientação semelhante à dos antifúngicos azólicos. A série azometínica, que apresenta a mesma atividade dual da série oxadiazolínica, embora com potências diferentes, não possui conformação adequada para a interação da forma proposta. Existem, no entanto, dados que indicam a possibilidade de interação de compostos no sítio catalítico da enzima de forma diferente em relação a compostos oxadiazolínicos e antifúngicos azólicos. / This study had as objective the study of Tridimensional Quantitative Structure-Activity Relationships, 3D QSAR, and the identification of potential targets for 5-nitro-heterocyclic compounds with azomethynic or oxadiazolynic structures presenting dual antifungal and anti-T. cruzi bioactivity, aiming the discovery of new compounds to be used as possible drug candidates or lead compounds. The studied compounds belong to four closely related series: AzoO series: 4-substituted 5-nitro-2-furfurylidene benzhydrazides; AzoS Series: 4-substituted 5-nitro-2-thiophylidene benzhydrazides; OxaO Series: 4-substituted 3-acetyl-oxadiazolyne 2,5-furfurylidene benzhydrazides; and OxaS Series: 4-substituted 3-acetyl-oxadiazolyne 2,5-thiophilydene benzhydrazides. The integrated use of tools belonging to the areas of medicinal chemistry, chemoinformatics, and bioinformatics allow for the characterization of catalytic cavities of proteins and the identification of physicochemical properties considered essential for the adequate interaction between ligand and target biomacromolecule. In addition to this, they also make possible the identification of targets through the comparison of chemical structures, with their associated physicochemical properties, of known ligands and their possible targets; this approach, known as virtual profiling, is based on the principle that molecules similar to known ligands of proteins can potentially interact with this same protein, with the similarity degree being determined not only by chemical structure but also through the distribution of physicochemical characteristics. The tools used in studies in silico also include the analysis of topological descriptors that can be used to analyse physicochemical characteristics considering their spacial distribution in relation to the chemical structure of a given molecule. The use of these descriptors makes possible the determination of the similarity degree between molecules or part of molecules (molecular fragments), and is the basis for methodologies of screening and characterization of the catalytic sites or proteins. Considering the proposed objective, studies were carried involving virtual profiling, analysis of molecular fragments based on physicochemical descriptors able to characterize the surface of ligands and proteins, characterization of the catalytic site of a possible target, and docking of the compounds to putative targets. The antifungal activity of compounds was determined and 3D QSAR results analysed, leading to the formulation of a hypothesis for the possible biological target for the azomethynic and oxadiazolynic compounds. Virtual profiling results pointed as possible targets the P450 enzymes CYP19A (aromatase), CYP3A4, CYP3A5, and CYP3A7. Although these enzymes are not directly involved with the currently tested biological activities for these compounds, there are studies reporting the interaction of azole antifungal compounds, whose target is CYP51 (14α-demethylase), with CYP19A and CYP3A4. Taken together with the analysis of physicochemical characteristics based on topological descriptors, and considering the duality of determined biological activities, it was concluded that the interaction of the studied compounds with CYP51 was possible since this enzyme is the target nor only for azole antifungals, but also for anti-T. cruzi compounds. Characterization studies of CYP51 catalytic cavity considering not only physicochemical properties descriptors but also stereochemical characteristics, associated to the results of 3D QSAR, confirmed the possibility of interaction of compounds from the oxadiazolynic series with CYP51, in an orientation similar to azole antifungals. The azomethynic series, that also presents the same dual biological activity though with different potency, does not present an adequate conformation for the ineraction proposed for the oxadiazolynic series; however, there are reports indicating the possibility of interaction of compounds in the catalytic site of the enzyme in a different way from that of antifungal azoles and the proposed interaction of oxadiazolynic compounds.