Search for bioactive compounds from medicinal plants used as antimalarials : the study of Momordica balsamina L.

Tese de doutoramento, Farmácia (Química Farmacêutica e Terapêutica), Universidade de Lisboa, Faculdade de Farmácia, 2010The main goal of this dissertation was to search for new antimalarial compounds from plants used in traditional medicine. For the purpose, the claimed antimalarial properties of fifty eight extracts from fifteen plants, used in traditional medicine against malaria and/or fever, mainly in Mozambique, were evaluated against the 3D7 Plasmodium falciparum strain. The highest activity was shown by Momordica balsamina L. (Cucurbitaceae), which was selected for further studies. Bioassay-guided fractionation of the methanol extract of M. balsamina led to the isolation of fourteen new cucurbitane-type triterpenoids, along with five known cucurbitacins and one megastigmane-type nor-isoprenoid. In order to obtain a higher homologous series of compounds required for structure-activity relationships, karavilagenin C and balsaminol F were esterified using several acylating agents, yielding twenty new derivatives, named karavoates A - R, triacetylbalsaminol F and tribenzoylbalsaminol F, respectively. The chemical structures of compounds were deduced from their physical and spectroscopic data (IR, UV, MS, HRMS, 1H and 13C NMR and 2D NMR experiments - COSY, HMQC, HMBC and NOESY experiments). Some of the new compounds feature unusual oxidation patterns, reported for the first time in cucurbitane triterpenoids from plant sources, such as at C-29 (balsaminagenins A, B, balsaminols A - D, and balsaminapentaol) and C-12 (cucurbalsaminols A, B, and C). Moreover, balsaminapentaol has a 23,24-diol system coupled with an exocyclic double bond in the side chain, never found before in cucurbitane-type triterpenoids. Compounds were evaluated for their antimalarial activity against the chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) P. falciparum strains. Most of them displayed antimalarial activity. Among the natural compounds, the glycoside derivatives and karavilagenin E revealed the highest activity against both strains of P. falciparum tested, displaying IC50 < 9 μM. A strong increase in the activity was found for the majority of alkanoyl ester derivatives of karavilagenin C and balsaminol F. Triacetylbalsaminol F, and karavoates B, D, and E displayed IC50 values similar to those obtained with chloroquine, particularly against the resistant strain (IC50 ≤ 0.6 μM). However, a significant decrease of activity was observed when both positions, C-3 and C-23 of the parent compounds, were esterified with aroyl or cinnamoyl chlorides, highlighting the influence of molecular esteric effects on the antimalarial activity. Moreover, the substitution pattern of ring B seems also to play an important role in the antiplasmodial activity of compounds. The preliminary toxicity toward human cells of compounds was also investigated on breast cancer cell line, and the selectivity index was calculated. Compounds were also evaluated for their ability as MDR reversers in both cancer cells and resistant bacteria strains. In cancer cells, the anti-MDR activity was carried out in human MDR1 gene-transfected mouse lymphoma cells, by flow cytometry. Most of the compounds exhibited a strong activity when compared with the one of the positive control, verapamil, in a non-toxic concentration. Karavilagenin C showed the strongest activity, at a low concentration. Structureactivity relationships will be discussed. The presence of free hydroxyl groups at C-3 and C-23 seems to be crucial for the reversing activity. Moreover, in the checkerboard model of combination chemotherapy, the interaction between doxorubicin and most of the compounds synergistically enhanced the effect of the anticancer drug. Some of the results obtained by flow cytometry were corroborated by using a real-time fluorometric method that employs ethidium bromide. Some of the compounds were also able to inhibit, significantly, the efflux of ethidium bromide by methicillinresistant Staphylococcus aureus highly resistant to oxacillin (MRSA COLoxa), and Enterococcus faecalis ATCC29212 strains. A good correlation between MRSA COLoxa reversal activity and the topological polar surface area of compounds was found. Momordica balsamina; Cucurbitane-type triterpenoids; Antimalarial activity; Plasmodium falciparum; Multidrug resistance; P-glycoprotein; MDR modulators.Esta dissertação teve como principal objectivo o isolamento de compostos com actividade antimalárica a partir de plantas usadas na medicina tradicional. Nesse sentido, foram avaliadas as propriedades antimaláricas de cinquenta e oito extractos provenientes de quinze plantas, usadas na sua maioria em Moçambique para o tratamento da malaria e/ou febres associadas, usando a estirpe sensível 3D7 de Plasmodium falciparum. A espécie Africana Momordica balsamina L. (Cucurbitaceae) demonstrou a melhor actividade, sendo escolhida para estudos posteriores. Do fraccionamento bio-guiado do extracto metanólico da espécie M. balsamina resultaram catorze novos compostos com o esqueleto do cucurbitano, juntamente com cinco compostos conhecidos com o mesmo esqueleto e um nor-isoprenóide com o esqueleto do megastigmano. De modo a obter um número de compostos que possibilitasse a realização de estudos de relação estrutura-actividade, os compostos karavilagenina C e balsaminol F, isolados em maiores quantidades da planta, foram esterificados. Do balsaminol F, por acilação em C-3/C-7/C-23 com anidrido acético ou cloreto de benzoílo, obtiveram-se os derivados triacilados, designados por triacetilbalsaminol F e tribenzoilbalsaminol F, respectivamente. Do mesmo modo, a karavilagenina C foi esterificada com vários anidridos/cloretos de ácido, originando dezoito novos compostos (monoésteres em C-23 e diésteres em C-3/C-23) designados de karavoatos A - R. Os compostos foram isolados utilizando técnicas cromatográficas (cromatografia em coluna, cromatografia preparativa em camada fina e cromatografia líquida de alta resolução). A caracterização estrutural foi estabelecida com base nas suas características físicas e dados espectroscópicos (IV, UV, MS, HRMS e RMN unidimensional- 1H, 13C, DEPT e bidimensional- COSY, HMQC, HMBC e NOESY). Alguns dos compostos apresentaram particularidades estruturais identificadas pela primeira vez em compostos com o esqueleto do cucurbitano, isolados a partir de plantas, nomeadamente no padrão de oxidação em C-29 (balsaminageninas A, B; balsaminois A - D e balsaminapentaol) e em C-12 (cucurbalsaminois A - C). O composto balsaminapentaol apresentou igualmente, na cadeia lateral, um sistema 23,24-diol vicinal a uma dupla ligação exocíclica, identificado pela primeira vez em compostos com o esqueleto do cucurbitano. A actividade antimalárica dos compostos obtidos foi avaliada, in vitro, em dois clones de P. falciparum, um sensível 3D7 e um resistente Dd2. A maioria dos compostos demonstrou actividade antimalárica. Dos compostos isolados, as melhores actividades foram observadas para os derivados glicosilados e karavilagenina E (IC50 < 9 μM). No que diz respeito aos ésteres, verificou-se um aumento pronunciado da actividade antimalárica para a maioria dos ésteres alifáticos da karavilagenina C e triacetilbalsaminol F. Com efeito, os derivados triacetilbalsaminol F e karavoatos B, D e E demonstraram uma actividade antimalárica (IC50 ≤ 0.6 μM) comparável à obtida com a cloroquina, principalmente na estirpe resistente de P. falciparum. Contudo, verificou-se que, quando ambas as posições C-3 e C-23 da karavilagenina C são substituídas por grupos aroílo ou cinamoílo, é observada uma diminuição significativa da actividade antimalárica. O mesmo sucedeu no caso do derivado tribenzoílado do balsaminol F. Estes resultados evidenciam a importância de efeitos estéreos na actividade antimalárica deste grupo de compostos. É também de salientar que o padrão de substituição no anel B parece influenciar a actividade. Foi realizado um ensaio preliminar de citotoxicidade em células humanas tumorais de mama (MCF-7). Os valores de IC50 obtidos neste ensaio permitiram o cálculo do índice de selectividade (razão entre a citotoxicidade e a actividade antimalárica) para todos os compostos. Os compostos foram também avaliados no que diz respeito à sua capacidade como reversores de multirresistência em células cancerígenas. Alguns compostos foram também testados em estirpes bacterianas resistentes. Deste modo, estudou-se a actividade anti-MDR em células de linfoma de rato transfectadas com o gene humano MDR1. Neste ensaio avaliou-se, por citometria de fluxo, a acumulação intracelular de rodamina-123, um substrato fluorescente análogo da doxorrubicina. A maioria dos compostos, numa concentração não citotóxica, demonstrou uma potente capacidade inibitória da actividade da glicoproteína-P quando comparada com a do verapamil, usado como controlo positivo. Dos compostos avaliados, a karavilagenina C demonstrou ser o mais activo, quando testado em concentrações baixas. O estudo realizado permitiu retirar algumas conclusões sobre a relação estrutura-actividade dos compostos. É de salientar a importância da presença de grupos hidroxilo livres nas posições C-3 e C-23 para a actividade. A lipofilia e a presença de grupos funcionais com capacidade para estabelecer ligações de hidrogénio foram também evidenciadas como características fundamentais para a actividade reversora da glicoproteína-P. Foram também avaliados os efeitos antiproliferativos in vitro de alguns destes triterpenos em combinação com a doxorrubicina. Todos os compostos, à excepção da balsaminagenina C que mostrou um efeito aditivo, demonstraram um efeito sinérgico sobre a actividade da doxorrubicina. Alguns dos resultados de actividade anti-MDR obtidos no ensaio com a rodamina-123 foram confirmados, utilizando um método fluorimétrico em tempo real que analisa a acumulação de brometo de etídio, um substrato fluorescente. A avaliação de alguns compostos como modeladores de resistência de estirpes bacterianas Gram-positivas e Gram-negativas foi igualmente realizada. Dos compostos testados, o balsaminol E, o balsaminosido A e a karavilagenina C inibiram significativamente o efluxo de brometo de etídio na estirpe de Staphylococcus aureus resistente à meticilina e adaptada à oxaciclina (MRSA COLoxa) e na de Enterococcus faecalis ATCC29212. É de salientar a correlação obtida entre a actividade reversora da estirpe MRSA COLoxa e a área de superfície polar dos compostos testados. Momordica balsamina; Triterpenos; Cucurbitano; Actividade antimalárica; Plasmodium falciparum, Multirresistência, Glicoproteína-P; Modeladores de multirresistência.This thesis was conducted at the Medicinal Chemistry Group of the Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculdade de Farmácia, Universidade de Lisboa. The financial support was provided by Fundação para a Ciência e a Tecnologia (SFH/BD/22321/2005)

Design, synthesis, and structure-activity relationship studies of dual Plasmodium falciparum phosphatidylinositol 4-kinase and cGMP-dependent protein kinase inhibitors

Malaria is a life-threatening disease caused by protists in the genus Plasmodium and transmitted by the female Anopheles mosquito. Amongst five species which infect humans, Plasmodium falciparum (Pf) causes the severest form of the disease. Although significant efforts have been made to reduce the overall impact of malaria in endemic regions, the ever emergence and continuous spread of parasite resistance to available chemotherapeutics, threatens to undermine advances made thus far. In addition, the current portfolio of drugs is non-effective in addressing chemoprotection, transmission blockade and relapse in P. vivax and P. ovale species. Thus, drugs targeting multiple stages of the parasite life cycle and of low risk to resistance, are highly desirable to support malaria elimination and/or eradication efforts. Considering the success of human kinase inhibitors as anti-cancer drugs and the identification of Plasmodium kinases as promising targets for malaria chemotherapy, this study aimed to optimize anti-plasmodium phosphatidylinositol 4-kinase (PI4K) and the cGMP-dependent protein kinase (PKG) inhibitors, based on two distinct chemotypes. Plasmodium PI4K and PKG are validated targets, each with the potential to deliver pan-stage active compounds with potentially moderate to low risk of resistance. Part 1 of this study focused on the repositioning of the oncological clinical Phase-1 mammalian target of rapamycin (mTOR) inhibitor, MLN0128, as a dual Plasmodium PI4K/PKG inhibitor for malaria. MLN0128 was identified by GlaxoSmithKline (GSK) Cellzome facility as a Plasmodium multi-kinase inhibitor with potent PI4K and PKG inhibitory activity. In this study, an in silico-guided structural modification strategy was undertaken towards optimizing dual Plasmodium kinase inhibition and anti-plasmodium activity while also mitigating potency against its oncological human target, mTOR and off-target PI4KIIIb (Figure 1). Arising from this work, analogues equipotent against both the chloroquine sensitive (PfNF54) and multi-drug resistant (PfK1) strains simultaneously targeting PI4K and PKG were identified. Docking studies using a PfPI4K homology model and a PvPKG crystal structure discerned the molecular features responsible for the high affinity of the inhibitors for these Plasmodium targets. Benzyl analogues containing a fluoro or chloro group at the meta or para positions displayed high anti-plasmodium activity with potent PvPI4K inhibition but weak PfPKG inhibition. Notable analogues included 7 (PfNF54 IC50 = 0.029 µM; PvPI4K IC50 = 0.007 µM; PfPKG IC50 > 2 µM) and 35 (PfNF54 IC50 = 0.086 µM; PvPI4K IC50 = 0.008 µM; PfPKG IC50 > 10 µM). Introduction of basic or pyridyl substituents proved important for dual Plasmodium kinase activity as exemplified by the active anti-plasmodium pyridyl analogues 44 (PfNF54 IC50 = 0.104 µM; PvPI4K IC50 = 0.004 µM; PfPKG IC50 = 0.834 µM) and 49 (PfNF54 IC50 = 0.189 µM; PvPI4K IC50 = 0.006 µM; PfPKG IC50 = 0.384 µM). In addition, the two compounds displayed low cytotoxicity against the Chinese Hamster Ovarian cell line, with a favorable selectivity index (CHO; SI > 100), low human ether-a-go-go-related gene (hERG) activity (IC50 > 10 µM) and high metabolic stability against human, rat, and mouse (H/R/M) liver microsomes (> 75% remaining after 30-min incubation). Selected compounds from the series also showed the potential for transmission blockade with specificity for stage IV/V gametocytes (IC50 100 µM). Compounds displayed potent PvPI4K inhibition but weak PfPKG inhibition (IC50 > 1 µM) in enzyme assays. Four compounds, including one sulfoxide analogue, displayed high stability when incubated with H/R/M liver microsomes in microsomal metabolic stability assays. These features also mitigated hERG activity as five analogues tested displayed an IC50 > 10 µM. Ultimately, a front-runner lead compound (86; GS1 16) with high biological activity and a good safety profile (PfNF54/PfK1 = 0.063/0.100 µM; PvPI4K IC50 = 0.003 µM; CHO SI > 793), optimal solubility (195 µM), favorable microsomal metabolic stability (H/R/M = 96/85/88%) and low affinity on the hERG-encoded potassium channel (IC50 = 44.80 µM), was identified for further progression

Medicinal chemistry approaches to malaria drug discovery

Tese de doutoramento, Farmácia (Química Farmacêutica e Terapêutica), Universidade de Lisboa, Faculdade de Farmácia, 2016Malaria remains a major burden to global public health, causing nearly 600,000 deaths annually. Efforts to control malaria are hampered by parasite drug resistance, insecticide resistance in mosquitoes, and the lack of an effective vaccine. However antimalarial drugs are a mainstay in efforts to control and eventually eradicate this disease, thus the discovery of new antimalarials is critical. Antimalarial drug discovery is especially challenging due to the unique biology of malaria parasites, the scarcity of tools for identifying new drug targets, and the poorly understood mechanisms of action of existing antimalarials. Therefore, this work describes the use of different medicinal chemistry approaches to address unmet needs in antimalarial drug discovery. Part of this process includes ensuring that sufficient drug leads are available to prime the drug discovery pipeline, particularly those with novel modes of action in order to limit issues of cross-resistance with existing drugs. The first approach described in this thesis consists on the phenotypic-based hit to lead optimization designed to explore the antimalarial potential of the 3-piperidin-4-yl-1H-indole. The hit compound was identified in a phenotypic screen of ~2 million compounds against asexual blood stage P. falciparum. Three series of analogs were synthesized following a reagentbased diversity approach, in a total of 38 compounds, and screened for their blood stage antimalarial activity. The SAR shows that 3-piperidin-4-yl-1H-indole is intolerant to most Npiperidinyl modifications. Out of the analogs synthesized, three were active (2.19, 2.20 and 2.29). Furthermore, the (4-(1H-indol-3-yl)piperidin-1-yl)(pyridin-3-yl)methanone (2.29) showed in vitro antimalarial activity (EC50 values ∼3 μM), no cross-resistance with chloroquine, selectivity for the parasite, and lead-like properties (cLogP 20). These results allow the prioritization of the phenylalanyl tRNA synthetase (FRS), histidyl tRNA synthetase (HRS), alanyl tRNA synthetase (ARS), and prolyl tRNA synthetase (PRS) as the top four enzymes for further exploration as drug targets in blood stage malaria. aaSA compound treatment of P. falciparum increased eIF2α phosphorylation at 100X concentration, inducing the amino acid starvation pathway through direct inhibition of the corresponding PfaaRS, with few exceptions. Moreover, analogs were also active in vitro against liver stage P. berghei, with > 99% parasite growth inhibition at the higher concentration of 10 μM. Thus, underscoring the potential of the aaRS family as an attractive novel class of antimalarial drug targets. To further explore cytoplasmic prolyl tRNA synthetase (cPRS) as an antimalarial target, which we have previously identified as the long sought biochemical target of the antimalarial halofuginone (HFG), novel HFG based inhibitors were designed to exploit additional ligandprotein interactions in the active site of cPRS, and may serve as lead compounds in the preclinical development of a mechanistically unique class of malaria drugs with activity against both liver and blood stage life cycle stages. Furthermore, we aimed to characterize the biology of cPRS inhibition and resulting amino acid starvation response. Understanding the enzymeinhibitor complex formed by the different types of inhibitors (HFG and L-ProSA) would further elucidate on the deferential effect observed on the amino acid starvation response in mammalian cells, despite targeting the same enzyme. Thus, a two-step proteomic approach to isolate the protein complex using immunoprecipitation followed by identification of its components using mass spectrometry is proposed. Despite not being able to completely establish the protocol, the results in MCF-7 cells are consistent with the model proposed, thus further work needs to be done towards increasing the amount of the enzyme-inhibitor complex isolated to meet the detection requirements of the techniques used. Finally, to address the problem of limited target identification and validation tools for novel antimalarial compounds, the third aim of this thesis investigates the use of fluorescently labeled small molecules as a novel target discovery approach in malaria drug discovery efforts. A new methodology, which originated from our efforts to synthetize MayaFluors in a one-pot, two-step approach via BODIPY-OTf intermediate, was developed to label drugs with the BODIPY fluorophore. The method allows for substitution of either one or both of the canonical fluorides on the BODIPY dye with alkoxy ligands, under mild conditions. We successfully labeled a group of small molecules. Of these, two known drugs ((+) JQ1 and hydroxychloroquine) were evaluated for their activity and cellular localization. In both cases the labeled drug presented comparable activity to the parent drug. Furthermore, the fluorescently labeled antimalarial displayed a subcellular staining pattern in mammalian cells that is consistent with accumulation in acidic vesicles in the cytoplasm. Moreover, the probe was also tested in Plasmodium falciparum cultures, where results show subcellular staining pattern that seems consistent with accumulation in the food vacuole. Despite the identified limitation concerning the solvent compatibility of the method, this approach allows direct labeling of hydroxylfunctionalized drugs, which we believe may have broad applications for rapid and specific imaging of elusive biological targets in living cells. Taken together, in this thesis multiple medicinal chemistry approaches are explored in an effort to identify novel antimalarial chemotypes that act on underexploited targets. Furthermore, these results present new opportunities for malaria drug discovery to aid efforts in malaria control and eventual eradication

Synthesis, Chemistry, Biological Evaluation And Structure-Activity Relationships Of Rac- And Chiral 6-Desmethyl-5Β –Hydroxy-D-Secoartemisinin And Analogs

The prevention and cure of malaria is difficult as it thrives under poor socio- and pharmacoeconomic conditions in third world, tropical and subtropical regions. It is a parasitic disease spread through the bite of an infected Anopheles mosquito. In humans, malaria can be caused by several Plasmodium species that include: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi. The natural product artemisinin is a sesquiterpene lactone with an endoperoxide bridge isolated from the Chinese annual herb qinghao (Artemisia annua L or sweet wormwood). The relatively stable endoperoxide bridge is essential for their antimalarial activity. The synthesis of rac-6-Desmethyl-5β–hydroxy-D-secoartemisinin (a 1,2,4-trioxane) afforded a starting point upon which to conduct heretofore unexplored structure-activity relationships (SAR) about the C5 position. Additionally, as the C4 position is quintessential, via a C4 radical, to issues related to the molecular details of the mechanism of action of artemisinin, C5 derivatives were hypothesized and synthesized in order to test the effect of stabilization of the C4 radical. Previous studies from the literature of separate enantiomeric peroxides revealed that chirality was unimportant for bioactivity. It was felt that this could not be true in all cases because clear bioorganic and biological evidence for the intermediacy of protein receptors exists. By separating two Mosher ester diastereomers of the title compound folloby saponification into the (3R, 5R, 5aR, 9aS) and (3S, 5S, 5aS, 9aR) enantiomeric alcohols. It was found that all activity was associated with the (3R, 5R, 5a R, 9aS) diastereomer. This mirror image overlays with the x-ray structure of the natural product, R-(+)-artemisinin (the 3 R trioxane). This is logical as the mechanism of action (MOA) of artemisinin and related 1,2,4-trioxanes appears to be related to protein binding folloby a Fe(II)-mediated C4 radical hypothesized to react covalently with a plasmodial SERCA cysteine moiety, blocking the SERCA Ca-channel. This radical is produced by an O1-O2/C3-C4 ring cleavage, suggesting that logical modifications at C5 would be expected to test this aspect of the proposed MOA and provide additional SAR to complement known modifications elsewhere in the backbone of the natural product

Design and synthesis of novel quinolones directed to the Plasmodium falciparum bc1 protein complex

Tese de Doutoramento, Química, Especialização em Química Orgânica, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2016Apesar dos intensos esforços para controlo da malária, esta doença parasítica milenar permanece como uma das doenças infeciosas conducentes a maiores taxas de mortalidade no mundo. É transmitida pela picada de um mosquito infectado do género Anopheles (apenas a fémea transmite a doença) e causada por um parasita do género Plasmodium. P. falciparum é a mais prevalente de entre as cinco estirpes de Plasmodium que afectam o homem e é também a mais mortal, sendo responsável por cerca de 90% do número total de óbitos por malária registados anualmente. Actualmente, a Organização Mundial de Saúde estima que cerca de 40% da população mundial está em risco de contrair a infecção e que, anualmente, cerca de 600 000 pessoas morrem devido a esta doença. O rápido desenvolvimento de resistência por parte de P. falciparum a pelo menos um dos fármacos antimaláricos convencionais de primeira linha e a terapias baseadas em artemisinina e seus derivados, estimula enormemente a comunidade científica e médica, convocando-a para a investigação e desenvolvimento de novos fármacos antimaláricos eficazes, seguros, acessíveis e fáceis de administrar, preferencialmente direcionados para novos alvos terapêuticos. A combinação farmacológica designada por Malarone® foi lançada pela GlaxoSmithKline para o tratamento e prevenção de malária causada por estirpes multiresistentes. Este medicamento resulta da combinação de dois fármacos antimaláricos: atovaquona e proguanil. A atovaquona é um competidor da ubiquinona, inibindo especificamente o complexo bc1 de P. falciparum através da sua ligação ao sítio Qo deste complexo. O complexo bc1 é essencialmente responsável pela transferência de electrões e translocação de protões ao longo da membrana mitocondrial, formando um potencial de membrana. A sua inibição leva à perda da função mitocondrial (relevante para o fornecimento de intermediários para a síntese de ATP e pirimidina), colapso do potencial electroquímico transmembranar e, finalmente, morte do parasita, validando este complexo como um potencial novo alvo terapêutico para o desenvolvimento de novos fármacos. Além de ser o local de ação da atovaquona, o sítio Qo é também conhecido como o local de ação da estigmatelina (composto natural) e da endoquina (um dos primeiros antimaláricos sintéticos). A endoquina serviu como modelo para o desenvolvimento de análogos (ELQ) que mantiveram o núcleo quinolínico na sua estrutura. Além da sua potencial utilização na quimioterapia da malária, a versatilidade do quimiotipo quinolona tem atraído intensa investigação, originando principalmente 4-oxo-quinolinas com propriedades farmacológicas melhoradas e com potencial aplicação terapêutica noutras áreas, como no tratamento de cancro, hepatite B, hepatite C, SIDA, herpes, infeções fúngicas ou tuberculose. A utilização das quinolonas como agentes antibacterianos teve início em 1963 com a descoberta do ácido nalidíxico. Muitos outros derivados quinolínicos com propriedades farmacológicas foram entretanto sintetizados, destacando-se as fluoroquinolonas norfloxacina, ciprofloxacina e levofloxacina. Nos últimos anos têm surgidos publicações que reportam uma grande variedade de compostos 4-oxo-quinolínicos com grupos éster em posição 3 que demonstram atividade antimalárica em concentrações que se situam na gama nanomolar baixa, atuando por inibição do complexo bc1 plasmodial. No entanto, devido à existência de algumas fragilidades nestes compostos, de que se destacam a fraca solubilidade em meio aquoso e baixa biodisponibilidade oral, esta classe de compostos requer optimização. O projecto de investigação descrito nesta tese foca-se no desenho, síntese e estudo estrutural de uma biblioteca de novas 3-etiléster-4-oxo-quinolinas (quinolonas), introduzindo diversidade química, essencialmente, nas posições 6 e/ou 7 da quinolona. Estudos de docking realizados in silico no sítio Qo do complexo bc1 (de levedura - yeast) sugerem um papel importante dos resíduos His182 e Glu272 no reconhecimento de potentes inibidores. Os mesmos estudos também indicam a importância da presença do grupo carbonilo e do grupo N-H do núcleo quinolónico na formação de ligações por pontes de hidrogénio com os resíduos proteícos His182 e Glu272, respectivamente. Deste modo, a possibilidade de tautomerismo entre as formas 4-oxo-quinolina e 4-hidroxi-quinolina não deve ser negligenciada, no contexto do desenvolvimento desta classe para terapia antimalárica. Esta tese está organizada em 5 capítulos. No capítulo 1 faz-se uma apresentação sobre a malária, bem como sobre os fármacos antimaláricos existentes, seu modo de ação e alvos terapêuticos moleculares. Os fármacos que atuam por inibição do complexo bc1 plasmodial e o uso e síntese de quinolonas em química medicinal também são abordados nesta secção. No capítulo 2 são descritas as abordagens sintéticas e a otimização das condições reacionais seguidas no desenvolvimento de quinolonas selecionadas. De uma forma geral, ao longo deste projecto de doutoramento, as quinolonas foram sintetizadas utilizando a metodologia de Gould-Jacobs. De acordo com esta metodologia ocorre ciclização térmica de um composto α,β-insaturado, derivado de uma anilina. Alternativamente, em algumas situações recorreu-se a uma outra estratégia utilizando o oxicloreto de fósforo. No decorrer destas sínteses, mais especificamente na etapa de ciclização, por vezes obtiveram-se alguns produtos inesperados relacionados com o padrão de substituição aromática decorrente da ciclização térmica e com a possibilidade de tautomerismo ceto-enólico. A possibilidade de tautomerismo entre as formas 4-oxo-quinolina e 4-hidroxi-quinolina suscitou questões do ponto de vista mecanístico e estrutural que são aqui especialmente relevantes, tendo em conta a sua eventual repercussão no perfil farmacodinâmico dos compostos. Assim, foi conduzida uma investigação estrutural detalhada em alguns compostos, de forma a melhor compreender a atividade e reactividade química de derivados de quinolonas, descrita no capítulo 3. Os resultados obtidos com base em estudos de cristalografia de raio X, espectroscopia FTIR acoplada com isolamento em matriz criogénica e cálculos teóricos são apresentados e discutidos, e indicam que, em derivados quinolínicos com um grupo éster em posição 3, a forma enólica (4-hidroxi-quinolina) é mais favorável do que a forma ceto (4-oxo-quinolina). O tautomerismo poderá traduzir-se em alterações nas propriedades químicas e físicas dos compostos, com possíveis implicações nos perfis farmacocinéticos e farmacodinâmicos dos mesmos. Os compostos sintetizados neste projecto foram testados in vitro contra estirpes sensíveis e resistentes de P. falciparum. Os resultados destes estudos, juntamente com estudos de avaliação da lipofilicidade/solubilidade de alguns compostos, são descritos e discutidos no capítulo 4. Tendo em conta os resultados de atividade biológica obtidos para alguns compostos e o facto de alguns estudos demonstrarem que certas quinolonas conseguem ultrapassar, embora parcialmente, os fenómenos de resistência à atovaquona, neste projecto de investigação estudou-se a possibilidade de ligação de potenciais fármacos a sítios alternativos e diferentes do sítio Qo do complexo bc1. Assim, foram efectuados estudos de simulação por docking no sítio Qi do complexo bc1, estando os resultados também descritos e discutidos no capítulo 4. Adicionalmente, foram sintetizadas algumas quinolonas substituídas com grupos arilo nas posições 2 e 3 que foram testadas contra estirpes de Mycobacterium tuberculosis, o agente responsável pelo desenvolvimento da tuberculose, cuja infeção é muitas vezes concomitante com a infeção pelo parasita da malária. A descrição e discussão deste trabalho é apresentada no capítulo 5. Para finalizar, e numa secção à parte, serão apresentadas as conclusões e considerações finais sobre o trabalho desenvolvido bem como algumas perspectivas para trabalho futuro.Malaria remains one of the most deadly infectious diseases. The growing spread of resistance by P. falciparum, the most prevalent strain affecting mankind, against conventional antimalarial drugs, urges the search for novel chemotherapeutics directed to new targets. The approval of Malarone® (combination of atovaquone and proguanil) for treatment and prevention of multidrug resistant malaria validated the P. falciparum bc1 complex as target for new antimalarials. Inhibition of bc1 leads to loss of mitochondrial function (relevant to provide intermediates for pyrimidine and ATP synthesis), collapse of the transmembrane electrochemical potential and, ultimately, parasite death. The versatility of the quinolone chemotype has attracted intense research, yielding mainly 4-oxo-quinolines with improved pharmacological properties, targeting a variety of applications, e.g., cancer, hepatitis, HIV, tuberculosis or malaria. Recent publications report on the high activity of some quinolone 3-esters against the plasmodial bc1 protein complex. However, due to some liabilities (e.g. poor solubility and low oral bioavailability), this class requires optimization. The research described within this thesis focus on the design, synthesis, structure and properties of a library of new 4-oxo-quinoline 3-esters substituted at positions 6 and/or 7 of the quinolone pharmacophore. Chapter one presents an introduction to malaria and antimalarial drugs. Other topics in this chapter are the plasmodial bc1 protein complex, the use of quinolones as inhibitors of this drug target and the synthetic routes to quinolones. In chapter two, synthetic approaches and conditions followed in the preparation of our library of quinolones, are described. For a better understanding of chemical reactivity and activity, a detailed structural investigation was undertaken on selected compounds, described in chapter three. Compounds synthesized in this project have been tested in vitro against P. falciparum. The results are discussed in chapter four, jointly with studies of lipophilicity/solubility evaluation and docking simulations. Additionally, selected quinolones were prepared and tested against Mycobacterium tuberculosis (chapter five). Conclusions and prospects for future work are presented in a final section

Repositioning of astemizole for malaria

Malaria remains one of the most important parasitic infectious diseases as far as human suffering is concerned. With almost half of the world's population at risk, its burden is felt worldwide as seen by the high number of deaths recorded each year (405,000 in 2018: WHO World Malaria Report 2019). Unfortunately, over 90% of this mortality rate is recorded in Africa alone, with the highest risk being in children under the age of five (5) and pregnant women. Partly, this is due to the unfortunate spread of resistance to most drugs that were once effective and safe, including Artemisinins which form the basis of the current first-line regimen in the treatment of malaria. For this reason, it is crucial to invest research efforts using various approaches in the drug discovery arsenal to develop novel, and structurally diverse antimalarials with different modes of action. These new antimalarials should not only be able to circumvent resistance but need to be efficacious at different life cycle stages of the parasite (multi-stage activity). This Ph.D. project pursued a drug repositioning approach on Astemizole (AST, Figure 1), a second-generation antihistamine drug which was previously identified as an antimalarial agent by Chong et al., at the Johns Hopkins University School of Medicine through via a high-throughput screening (HTS) of diverse marketed drugs. AST was active against chloroquine-sensitive (CQ-S) and multi-drug resistant (MDR) laboratory strains of the human malaria parasite Plasmodium falciparum (P. falciparum) and demonstrated in vivo efficacy in two mouse infection models of malaria namely, P. Vinckei and P. Yoelii. However, in addition to its low solubility, AST possesses a serious and fatal cardiotoxicity risk, evidenced by its ability to potently inhibit the human ether-á-go-go-related gene (hERG) encoded potassium (K+) channels. This liability led to the withdrawal of AST in most countries during the late 1970's and it is still being discontinued for use in some countries to date

Metabolomic Investigation of Natural Antiplasmodial Agents

Malaria is one of the deadliest parasitic diseases that still plagues humanity in recent times. Despite being target of several eradication campaigns, the widespread presence of the multiple agents that cause this parasitosis, along with its increased adaptability into developing resistance to treatments, make this old ailment a challenge to modern medicine. Natural products with potential antiplasmodial activity are valuable to discover new therapeutic targets and shed light on new scaffolds that can be optimized into revolutionary treatments. In this thesis, metabolomics, a sensitive and robust approach that can describe the metabolites of the malaria parasite maintained in culture, was used to profile the effects of natural products on the parasite and hypothesize their mode of action. Before this could be done, workflow investigation and protocol optimization were carried out, including a study on metabolic extraction methods with state-of-the-art statistical algorithms. Further exploration revealed that time and complexity are detrimental to repeatability and robustness of the results, so a method with a single washing step followed by methanolic 90% extraction was used and analyzed through 1HNMR and/or LC-MS, depending on sample availability. Five individual cases were studied in collaboration with the Department of Biochemistry and Molecular Biology and the Huck Center for Malaria Research at Penn State (The Pennsylvania State University), which allowed the use of their inhouse MS system and database for targeted metabolite annotation. First, an exploration of plant extracts and fractions were studied in parallel to the known active antiplasmodial compounds present in those plants to assess how early in plant screening a potential mode of action can be hypothesized. Results show that extract effects can already be distinguished from a control, though fractions with active compounds separate more clearly. The more purified the fraction, the bigger the correlation in mode of action with the isolated natural compounds, possibly due to the lack of matrix effects and interactions, which shows that metabolomics can be introduced early in bioassay-guided fractionation in plant studies. Second, two of the most important plants in traditional medicine against malaria were studied, Artemisia afra and A. annua, to investigate their phenolic content, presence or absence of artemisinin and correlate the composition with the metabolomic results on a synchronized malaria culture. Results indicate a correlation between activity and artemisinin abundance in these extracts, with A. annua presenting a similar parasitic profile to artemisinin whereas A. afra, despite trace amounts of this compound, differs significantly. A. afra affects various unspecific metabolites but significantly changes myo-inositol distinctly from A. annua and artemisinin, which clearly target redox mechanisms. Lastly, three independent studies aimed to investigate three natural scaffolds for their mode of action: alkyl cyclohexenones compounds named poupartones, ellagic acid and derivatives, and a mixture of triterpene esters. Poupartones showed to interfere with hemoglobin metabolism, DNA and RNA synthesis and redox management systems which correlated to their potential to participate in nucleophilic additions that establish covalent bonds with proteins and generate radical oxygen species, an effective yet not specific type of activity. Our studies on ellagic acid and derivatives support literature data and point to the parasite’s digestive vacuole as the site of action of these compounds. Changes in hemoglobin metabolism and redox metabolites suggest possible effects on plasmepsins, enzymes that act early in hemoglobin breakdown, and on glutathione metabolism, essential to maintaining a balanced organelle. Lastly, a mixture of 8 triterpenic esters seems to affect pyrimidine synthesis and amino acid metabolism through N-carbamoyl-L-aspartate, though it is unclear exactly how. Metabolomics is a hypothesis generating approach that gives a snapshot of the effects of innovative natural compounds on the malaria parasite in order to accurately guide antimalarial drug discovery.METNATPA

Application of computer-aided drug design for identification of P. falciparum inhibitors

Malaria is a millennia-old disease with the first recorded cases dating back to 2700 BC found in Chinese medical records, and later in other civilizations. It has claimed human lives to such an extent that there are a notable associated socio-economic consequences. Currently, according to the World Health Organization (WHO), Africa holds the highest disease burden with 94% of deaths and 82% of cases with P. falciparum having ~100% prevalence. Chemotherapy, such as artemisinin combination therapy, has been and continues to be the work horse in the fight against the disease, together with seasonal malaria chemoprevention and the use of insecticides. Natural products such as quinine and artemisinin are particularly important in terms of their antimalarial activity. The emphasis in current chemotherapy research is the need for time and cost-effective workflows focussed on new mechanisms of action (MoAs) covering the target candidate profiles (TCPs). Despite a decline in cases over the past decades with, countries increasingly becoming certified malaria free, a stalling trend has been observed in the past five years resulting in missing the 2020 Global Technical Strategy (GTS) milestones. With no effective vaccine, a reduction in funding, slower drug approval than resistance emergence from resistant and invasive vectors, and threats in diagnosis with the pfhrp2/3 gene deletion, malaria remains a major health concern. Motivated by these reasons, the primary aim of this work was a contribution to the antimalarial pipeline through in silico approaches focusing on P. falciparum. We first intended an exploration of malarial targets through a proteome scale screening on 36 targets using multiple metrics to account for the multi-objective nature of drug discovery. The continuous growth of structural data offers the ideal scenario for mining new MoAs covering antimalarials TCPs. This was combined with a repurposing strategy using a set of orally available FDA approved drugs. Further, use was made of time- and cost-effective strategies combining QVina-W efficiency metrics that integrate molecular properties, GRIM rescoring for molecular interactions and a hydrogen mass repartitioning (HMR) molecular dynamics (MD) scheme for accelerated development of antimalarials in the context of resistance. This pipeline further integrates a complex ranking for better drug-target selectivity, and normalization strategies to overcome docking scoring function bias. The different metrics, ranking, normalization strategies and their combinations were first assessed using their mean ranking error (MRE). A version combining all metrics was used to select 36 unique protein-ligand complexes, assessed in MD, with the final retention of 25. From the 16 in vitro tested hits of the 25, fingolimod, abiraterone, prazosin, and terazosin showed antiplasmodial activity with IC50 2.21, 3.37, 16.67 and 34.72 μM respectively and of these, only fingolimod was found to be not safe with respect to human cell viability. These compounds were predicted active on different molecular targets, abiraterone was predicted to interact with a putative liver-stage essential target, hence promising as a transmission-blocking agent. The pipeline had a promising 25% hit rate considering the proteome-scale and use of cost-effective approaches. Secondly, we focused on Plasmodium falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase (PfDXR) using a more extensive screening pipeline to overcome some of the current in silico screening limitations. Starting from the ZINC lead-like library of ~3M, hierarchical ligand-based virtual screening (LBVS) and structure-based virtual screening (SBVS) approaches with molecular docking and re-scoring using eleven scoring functions (SFs) were used. Later ranking with an exponential consensus strategy was included. Selected hits were further assessed through Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA), advanced MD sampling in a ligand pulling simulations and (Weighted Histogram Analysis Method) WHAM analysis for umbrella sampling (US) to derive binding free energies. Four leads had better predicted affinities in US than LC5, a 280 nM potent PfDXR inhibitor with ZINC000050633276 showing a promising binding of -20.43 kcal/mol. As shown with fosmidomycin, DXR inhibition offers fast acting compounds fulfilling antimalarials TCP1. Yet, fosmidomycin has a high polarity causing its short half-life and hampering its clinical use. These leads scaffolds are different from fosmidomycin and hence may offer better pharmacokinetic and pharmacodynamic properties and may also be promising for lead optimization. A combined analysis of residues’ contributions to the free energy of binding in MM-PBSA and to steered molecular dynamics (SMD) Fmax indicated GLU233, CYS268, SER270, TRP296, and HIS341 as exploitable for compound optimization. Finally, we updated the SANCDB library with new NPs and their commercially available analogs as a solution to NP availability. The library is extended to 1005 compounds from its initial 600 compounds and the database is integrated to Mcule and Molport APIs for analogs automatic update. The new set may contribute to virtual screening and to antimalarials as the most effective ones have NP origin.Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 202

Synthesis, characterisation and in vitro evaluation of hybrid compounds containing ferrocene and nitrogen-containing bisphosphonates

Abstract: Ferrocene derivatives, namely, 4-ferrocenyl-4-oxo-butanoic acid (F1), 4-ferrocenylbutanoic acid (F2), ferrocenylmethyleneamine (F5) and ferrocenylethyleneamine (F6), were synthesized. The aminobisphosphonic acids, Alendronate ((4-amino-1-hydroxy-1-phosphobutyl) phosphonate), B1, (5-amino-1-hydroxy-1-phosphopentyl) phosphonate, B2, and neridronate ((6-amino-1-hydroxy-1-phosphohexyl) phosphonate), B3, tetraethylvinylidenephosphonate, B4, and tetraethyl-3-aminopropane-1-1-bisphosphonate, B5 were also synthesized. Various ferrocene and bisphosphonates derivates were then hybridized via amine and amide bond formation to get a series of ferrocene-bisphosphonate hybrid compounds (H1-H8) in 55-83% yield. H1-H3 were synthesized by reductive amination of ferrocene carboxaldehyde, F4 with aminobisphosphonic acids, B1, B2 and B3 respectively. Hybrids H4-H6, were obtained by direct coupling of ferrocenecarboxylic acid, F3, 4-ferrocenylbutanoic acid, F2 and 4-ferrocenyl-4-oxo-butanoic acid, F1, respectively with tetraethyl-3-aminopropane-1-1-bisphosphonate, B5, using a 50 % solution of propylphosphonic anhydride (T3P) in ethyl acetate as a coupling agent. H7a was prepared from ferrocenylmethyleneamine (F5) and tetraethylvinylidenebisphosphonate (B4) in a Michael addition type reaction, while H8 was obtained by reductive amination of ferrocene carboxaldehyde, F4, with tetraethyl-3-aminopropane-1-1-bisphosphonate, B5. All synthesized compounds were characterised by a combination of 1H, 31P{1H} and 13C{1H} nuclear magnetic resonance (NMR) spectroscopy, fourier transform-infrared (FT-IR) and high-resolution mass spectrometry (HRMS)...D.Phil. (Chemistry