Magister Pharmaceuticae - MPharm / Plasmodium falciparum malaria continues to be a worldwide health problem, especially in developing countries in Africa and is responsible for over a million fatalities per annum. Chloroquine (CQ) is low-cost, safe and was the mainstay aminoquinoline derived chemotherapeutic agent that has been used for many years against blood-stage malaria. However, today the control of malaria has been complicated by increased resistance of the malaria parasite to existing antimalarial agents such as CQ. The primary cause of resistance is mutation in a putative ATP-powered multidrug efflux pump known as the p-glycoprotein (pGP) pump, and point mutation in P. falciparum CQ resistance transporter (PfCRT) protein. These mutations are responsible for the reduced accumulation of CQ at its primary site of action, the acidic digestive food vacuole of the parasite.To overcome the challenges of CQ resistance in P. falciparum, chemosensitiser offer an attractive approach. Chemosensitisers or reversal agents are structurally diverse molecules that are known to reverse CQ resistance by inhibiting the pGP efflux pump and/or the PfCRT protein associated with CQ export from the digestive vacuole in CQ resistant parasites. Chemosensitisers include the well-studied calcium channel blocker verapamil and antihistaminic agent chlorpheniramine. These drugs have little or no inherent antimalarial activity but have shown to reverse CQ resistance in P. falciparum when co-administered with CQ. Because of the channel blocking abilities of pentacycloundecylamines (PCUs) such as NGP1-01, it is postulated that these agents may act as chemosensitisers and circumvent the resistance of the Plasmodium parasite against CQ. Therefore as a proof of concept we conducted an experiment using CQ co- administered with different concentrations of NGP1-01 to evaluate the ability of NGP1-01 to act as a chemosensitiser.Herein, we report the ability of NGP1-01, the prototype pentacycloundecylamine (PCU), to reverse CQ resistance (> 50 %) and act as a chemosensitiser. NGP1-01 alone exhibited very low intrinsic antimalarial activity against both the resistant and sensitive strain (> 2000 nM), with no toxicity to the parasite detected at 10 µM. A statistically significant (p < 0.05) dose dependent shift was seen in the CQ IC50 values at both 1 µM and 10 µM concentration of co-administeredNGP1-01 against the resistant strain. Based on this finding we set out to synthesise a series of novel agents comprising of a PCU moiety as the reversal agent (RA) conjugated to a CQ-like aminoquinoline (AM) molecule and evaluate the potential of these PCU-AM derivatives as antimalarial- and/or reversed CQ agents. As recently shown by Peyton et al., (2012), the conjugation of a CQ-like molecule with a RA such as the chemosensitiser imipramine and derivatives thereof is a viable strategy to reverse CQ resistance in multidrug-resistant P. falciparum. The novel compounds were obtained by amination and reductive amination reactions. The synthetic procedures involved the conjugation of the Cookson’s diketone with different tethered 4-aminoquinoline moieties to yield the respective carbinolamines and the subsequent imines. This was followed by a transannular cyclisation using sodium cyanoborohydride as reducing agent to yield the desired PCU-AM derivatives. The CQ-like AMderivatives were obtained using a novel microwave (MW) irradiation method. Structure elucidation was done by utilising 1H- and 13C NMR spectroscopy as well as IR absorption spectrophotometry and mass spectrometry. Five PCU-AM reversed CQ derivatives were successfully synthesised and showed significant in vitro antimalarial activity against the CQ sensitive strain (NF54). PCU-AM derivatives 1.1 – 1.4 showed antimalarial IC50 values in the ranges of 3.74 – 17.6 ng/mL and 27.6 – 253.5 ng/mL against the CQ-sensitive (NF54) and CQ-resistant strains (Dd2) of Plasmodium falciparum, respectively. Compound 1.1 presented with the highest antimalarial activity against both strains and was found to be 5 fold more active against the resistant strain than CQ. The reversed CQ approach resulted in improved resistance reversal and a significantly lower concentration PCU was required compared to NGP1-01 and CQ in combination. This may be attributed to the improved ability of compound 1.1 to actively block the pGP pump and/or the increased permeability thereof because of the lipophilic aza-PCU moiety. Compound 1.1 also showed the lowest RMI value confirming that this compound has the best potential to act as a reversed CQ agent in the series. Cytotoxicity IC50 values observed for compounds 1.1 – 1.4 were in the low micromolar concentrations (2.39 – 9.54 µM) indicating selectivity towards P. falciparum (SI = 149 – 2549) and low toxicity compared to the cytotoxic agent emetine (IC50 = 0.061 µM).These results indicate that PCU channel blockers and PCU-AM derived conjugates can be utilised as lead molecules for further optimisation and development to enhance their therapeuticpotential as reversal agents and reversed CQ compounds.

No / A series of 19 huprines has been evaluated for their activity against cultured bloodstream forms of Trypanosoma brucei and Plasmodium falciparum. Moreover, cytotoxicity against rat myoblast L6 cells was assessed for selected huprines. All the tested huprines are moderately potent and selective trypanocidal agents, exhibiting IC50 values against T. brucei in the submicromolar to low micromolar range and selectivity indices for T. brucei over L6 cells of approximately 15, thus constituting interesting trypanocidal lead compounds. Two of these huprines were also found to be active against a chloroquine-resistant strain of P. falciparum, thus emerging as interesting trypanocidal–antiplasmodial dual acting compounds, but they exhibited little selectivity for P. falciparum over L6 cells.

Yes / Current drugs against human African trypanosomiasis (HAT) suffer from several serious drawbacks. The search for novel, effective, brain permeable, safe, and inexpensive antitrypanosomal compounds is therefore an urgent need. We have recently reported that the 4-aminoquinoline derivative huprine Y, developed in our group as an anticholinesterasic agent, exhibits a submicromolar potency against Trypanosoma brucei and that its homo- and hetero-dimerization can result in to up to three-fold increased potency and selectivity. As an alternative strategy towards more potent smaller molecule anti-HAT agents, we have explored the introduction of ω-cyanoalkyl, ω-aminoalkyl, or ω-guanidinoalkyl chains at the primary amino group of huprine or the simplified 4-aminoquinoline analogue tacrine. Here, we describe the evaluation of a small in-house library and a second generation of newly synthesized derivatives, which has led to the identification of 13 side chain modified 4-aminoquinoline derivatives with submicromolar potencies against T. brucei. Among these compounds, the guanidinononyltacrine analogue 15e exhibits a 5-fold increased antitrypanosomal potency, 10-fold increased selectivity, and 100-fold decreased anticholinesterasic activity relative to the parent huprine Y. Its biological profile, lower molecular weight relative to dimeric compounds, reduced lipophilicity, and ease of synthesis, make it an interesting anti-HAT lead, amenable to further optimization to eliminate its remaining anticholinesterasic activity. / Wellcome Trust

Diferent classes of antimalarial drugs have been used to treat malaria, but the emergence of resistant strains of Plasmodium these drugs have decreased their efficiency, causing serious public health problem in tropical areas of the world. In this context, we carried out the preparation and antimalarial evaluation of eleven organic compounds: 7-Cl-MAQ, MAQPZ, 7-Cl-MAQPZ, 2-CF3-MAQ, 7-CF3-MAQ, 7-CF3-MAQPZ, 2-CF3-MAQPZ, 8-Cl-MAQ, 7-Cl-BAQ, 7-CF3-BAQ, 8-Cl-BAQ. These were obtained from the same synthetic strategy, via nucleophilic aromatic substitution reaction between 4-chloroquinoline derivatives and diethylenetriamine or 1,2-(aminoethyl) piperazine, reaching moderated to high yields. These compounds were designed based on the molecular structure of chloroquine, a classic antimalarial drug. As for antimalarial activity, only four substances (7-Cl-MAQ, BAQ, 2-CF3-MAQ and 7-CF3-MAQ) were evaluated. Note that the 7-Cl-MAQ was more active in in vitro and in vivo that the BAQ. The same behavior occurred in cytotoxicity assays, with higher values than the BAQ. In in vivo tests, the monoquinolínico derivative (7-Cl-MAQ) significantly reduced parasitemia. Have the 2-CF3-MAQ compounds and 7-CF3-MAQ, were active in vitro, but were inactive in vivo. Keywords: 4-aminoquinolinic derivatives, antimalarials, chloroquine. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Várias classes de antimaláricos têm sido utilizadas para o tratamento da malária, porém o surgimento de cepas de Plasmodium resistentes a esses fármacos têm diminuído suas eficácias, provocando graves problemas de saúde pública em áreas tropicais do mundo. Nesse contexto, realizou-se a preparação, caracterização e avaliação antimalárica de onze derivados aminoquinolínicos: 7-Cl-MAQ, MAQ, MAQPZ, 7-Cl-MAQPZ, 2-CF3-MAQ, 7-CF3-MAQ, 7-CF3-MAQPZ, 2-CF3-MAQPZ, 8-Cl-MAQ, 7-Cl-BAQ, 7-CF3-BAQ, 8-Cl-BAQ. Estes foram obtidos através da mesma estratégia sintética, via reação de substituição nucleofílica aromática, a partir da reação entre derivados de 4-cloroquinolinas com dietilenotriamina ou 1,2-(aminoetil)piperazina, chegando a rendimentos moderados altos a. Estes compostos foram elaborados baseados na estrutura molecular da cloroquina, um clássico fármaco antimalárico. Quanto à atividade antimalárica, apenas quatro substâncias (7-Cl-MAQ, BAQ, 2-CF3-MAQ e 7-CF3-MAQ) foram avaliadas. Vale destacar que a 7-Cl-MAQ foi mais ativa nos testes in vitro e in vivo que a BAQ. O mesmo comportamento ocorreu nos ensaios de citotoxicidade, apresentando valores maiores que a BAQ. Nos testes in vivo, o derivado monoquinolínico (7-Cl-MAQ) reduziu significativamente a parasitemia. Já os compostos 2-CF3-MAQ e 7-CF3-MAQ, mostraram-se ativos in vitro, porém foram inativos in vivo.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-06-21T15:25:14Z No. of bitstreams: 1 lucianamariaribeiroantinarelli.pdf: 3736513 bytes, checksum: 7a35a693236ba9e982e630b172b6f3cf (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-08-07T19:10:52Z (GMT) No. of bitstreams: 1 lucianamariaribeiroantinarelli.pdf: 3736513 bytes, checksum: 7a35a693236ba9e982e630b172b6f3cf (MD5) / Made available in DSpace on 2017-08-07T19:10:52Z (GMT). No. of bitstreams: 1 lucianamariaribeiroantinarelli.pdf: 3736513 bytes, checksum: 7a35a693236ba9e982e630b172b6f3cf (MD5) Previous issue date: 2013-02-28 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A quimioterapia atual para as leishmanioses está longe do ideal devido a uma série de problemas como alto custo e elevada toxicidade. Assim, existe uma necessidade imediata de obtenção de novos fármacos para o tratamento da doença. Neste contexto, derivados de quinolina têm demonstrado atividade leishmanicida promissora. Neste trabalho, foi avaliada a atividade leishmanicida de duas séries distintas de quinolinas: seis compostos derivados de 4-aminoquinolinas (4-AMQ) e seus híbridos com esteroide e nove derivados da amodiaquina (AQ). O efeito dos compostos foi testado em promastigotas e amastigotas de Leishmania sp e em macrófagos peritoneais de camundongos. Os resultados mostraram que a conjugação de 4-AMQ ao esteroide resultou em aumento significativo da atividade principalmente para formas promastigotas e amastigotas de L. major. O composto 6 foi o mais ativo em amastigota de L. major (CI50 de 1,9 µM), sendo três vezes mais efetivo do que a miltefosina. Demonstrou-se neste trabalho que a conjugação de dois grupos de grande aplicação biológica, quinolina e esteroide, pode ser uma estratégia interessante para o desenvolvimento racional de novos fármacos. Em relação aos derivados da AQ, observou-se que a grande maioria dos compostos foram ativos em promastigotas e amastigotas de Leishmania sp. Dentre os nove compostos avaliados, seis foram mais ativos em amastigotas de L. braziliensis do que o protótipo AQ e miltefosina. O composto 8 (CI50 de 0,4 µM) foi cerca de quatorze vezes mais ativo em amastigota do que a miltefosina. A maioria dos derivados de 4-AMQ e AQ foram mais seletivos e específicos para amastigotas. A atividade em potencial dos compostos avaliados pode ser considerada um importante avanço no estudo desta classe de derivados, visando-se o desenvolvimento de novos fármacos leishmanicidas mais eficazes, seletivos e não tóxicos para o hospedeiro humano. / Current chemotherapy for leishmaniasis is far from ideal due to a number of problems such as high cost and high toxicity. Thus, there is an immediate need to obtain new drugs for the treatment the disease. In this context, quinoline derivatives have shown promising antileishmanial activity. In this study, we evaluated the activity of two distinct series of quinolines, six 4-aminoquinolines (4-AMQ) derivatives and their hybrids with steroid and nine amodiaquine (AQ) derivatives in different Leishmania species. Effect of the compounds was assayed against Leishmania promastigotes and amastigotes and mouse peritoneal macrophages. Results showed that the combination of 4-AMQ to the steroid resulted in a significant increase in activity mainly for L. major promastigotes and amastigotes forms. Compound 6 was the most active against L. major amastigotes with IC50 of 1.9 µM, being three times more effective than miltefosine. It was demonstrated in this work that the combination of two groups with large biological application as quinoline derivatives and steroids may be an interesting strategy for the rational development of new drugs. Regarding to the AQ derivatives, it was observed that all compounds were most active against Leishmania promastigote and amastigote forms. Among the nine compounds evaluated, six were more active against L. braziliensis amastigotes than the prototype AQ and miltefosine. Compound 8 with IC50 of 0.4 µM was about fourteen times more active than miltefosine. In general, the majority of 4-AMQ and AQ derivatives are more selective and specific for amastigotes of Leishmania sp, forms clinically relevant. Potential activity of the compounds evaluated can be considered an important advance in the study of this class of derivatives in order to develop new leishmanicidal drugs more effective, selective and nontoxic to the human host.

We have synthesized a series of dimers of (+)-(7R,11R)-huprine Y and evaluated their activity against Trypanosoma brucei, Plasmodium falciparum, rat myoblast L6 cells and human acetylcholinesterase (hAChE), and their brain permeability. Most dimers have more potent and selective trypanocidal activity than huprine Y and are brain permeable, but they are devoid of antimalarial activity and remain active against hAChE. Lead optimization will focus on identifying compounds with a more favourable trypanocidal/anticholinesterase activity ratio.

NRH quinone oxidoreductase 2 (NQO2) is regarded as a mammalian Phase I detoxifying enzyme responsible for reducing quinones to hydroquinones. NQO2 is highly expressed in different types of cancer such as breast and prostate cancer suggesting its participatory role in the progression of these diseases. A potential reason for this is that NQO2 has the ability to modulate the stability of cyclin D1 and activity of NF-ÃŽÂoB and it has been shown that inhibition of NQO2, either genetically or pharmacologically, can alter the pattern of proliferation of cancer cells. However, the biological roles of NQO2 in cancer progression are still ambiguous and need further investigation. A panel of seven ovarian cancer cell lines (OVCs) were screened for the presence and functionality of NQO2. SKOV-3 and TOV-112D cells expressing comparatively the highest and lowest levels of NQO2 were stably transduced to silence and overexpress NQO2 respectively. Pharmacological inhibition was achieved using resveratrol or a series of novel 4-aminoquinolines synthesised in-house. Cell proliferation was monitored by cell counting and clonogenic assays. Flow cytometric analysis was used to determine cell cycle distribution and levels of ROS following modulation of NQO2 function. The expression of cell cycle regulatory markers was determined by Western blot. The contributory roles of NQO2 in determining the cytotoxicity of Adriamycin (ADR) towards OVCs was investigated using MTT assay together with evaluation of P-gp expression and basal ROS levels. In the OVCs panel, NQO2 protein levels and enzymatic activity showed an excellent correlation; with activity varying 36-fold between the cell lines. The sensitivity of OVCs to CB1954 was significantly increased when combined with the NRH-like co-factor, EP0152R. This supports the notion that NQO2 mediates the toxicity of CB1954, which is further confirmed by the strong correlation between cellular NQO2 activity and the responsiveness of the OVC cell lines to CB1954. Hydrazone quinolines showed the highest inhibitiory potency against NQO2 in SKOV-3 when compared to the typical and in-house synthesised quinolines inhibitors. NQO2-overexpressing TOV-112D cells showed more aggressive growth pattern and higher capacity to form colonies than wild-type cells. This was consistently associated with an enhancement in the progression of cells through cell cycle phases and significant reduction in Rb expression. A reduction in ROS levels in NQO2-OE cells may also explain this enhancement in cell growth. Overexpressing NQO2 also resulted in destabilisation of CDK4 and cyclin D1 with significant reduction in their expression levels, and concomitant increase in p-cyclin D1 (Thr286). The involvement of NQO2 in controlling cyclin D1 turnover is also confirmed in SKOV-3 cells when genetic silencing of NQO2 was accompanied by significant reduction in p-cyclin D1 and subsequent stabilisation of cyclin D1 levels. In spite of this, no alterations in the growth pattern of SKOV-3 cells were observed highlighting the impact of cell type on the variations in cellular responses. The role of NQO2 in determining the toxicity of ADR treatment was not proved in OVC cells. This was despite that modulation of NQO2 levels caused significant changes in P-gp expression. The intracellular basal levels of ROS was found to affect the responsiveness of OVCs to ADR as demonstrated when treating SKOV-3 with resveratrol was accompanied by significant increase in ROS levels and concomitant enhancement in the cells’ response to ADR. In conclusion, NQO2 can profoundly alter the proliferation characteristics of OVCs and is a potential therapeutic target for the treatment of this disease. However, the biological functions of NQO2 and its contributory roles in particular pathways are varied among different types of cancer -in other words- are highly dependent on cancer type.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-05-25T14:41:06Z No. of bitstreams: 1 isabeladeoliveirasouza.pdf: 8840752 bytes, checksum: de1f7daf9c0f6025ccc57475de168c79 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-05-25T14:48:39Z (GMT) No. of bitstreams: 1 isabeladeoliveirasouza.pdf: 8840752 bytes, checksum: de1f7daf9c0f6025ccc57475de168c79 (MD5) / Made available in DSpace on 2017-05-25T14:48:39Z (GMT). No. of bitstreams: 1 isabeladeoliveirasouza.pdf: 8840752 bytes, checksum: de1f7daf9c0f6025ccc57475de168c79 (MD5) Previous issue date: 2017-03-07 / Este trabalho mostra a síntese de 26 derivados aminoquinolínicos e suas avaliações em protozoários. Para uma melhor apresentação do projeto este foi dividido em três séries: a primeira está relacionada à síntese de hidrazonas e a segunda à síntese de bases de Schiff. Os dois núcleos bioativos foram conjugados envolvendo o conceito de hibridação molecular. Já a terceira série envolveu a síntese de complexos de prata, a partir dos ligantes 4-aminoquinolínicos. Todos os compostos foram devidamente caracterizados utilizando técnicas espectroscópicas que permitiram a identificação dos mesmos. Os compostos sintetizados tiveram seu potencial antiparasitário avaliado em parasitos do gênero Leishmania e Plasmodium, que são responsáveis pelas doenças Leishmanioses e Malária, respectivamente. Além disto, tiveram sua toxidez avaliada em células de mamíferos, tendo sido escolhidos macrófagos peritoneais de camundongos. Em relação à atividade leishmanicida, parte dos compostos foram ativos em Leishmania sp, com destaque para o composto 1a em formas amastigotas de L. amazonensis (CI50 de 8,1 µM), resultado próximo ao fármaco utilizado como referência, Miltefosina (CI50 de 4,15µM). A maioria dos compostos não apresentou citotoxicidade expressiva para os macrófagos, com exceção dos compostos 1b, 1f e 2d. Os testes em Plasmodium ainda estão em fase de análise. Os resultados aqui apresentados confirmam o potencial biológico de derivados aminoquinolinas e estimulam a continuidade dos estudos com esta classe de moléculas para o tratamento de doenças parasitárias. / This work demonstrates the synthesis of 26 aminoquinolinic derivatives and their evaluation in protozoa. For a better display, the project was divided into three series: the first is related to hydrazone synthesis and the second to synthesis of Schiff bases. The two bioactive nuclei were conjugated involving the concept of molecular hybridization. The third series involved the synthesis of silver complexes from the 4-aminoquinoline ligands. All components were properly characterized using spectroscopic techniques that allowed their identification. The synthesized compounds had their antiparasitic potential evaluated in parasites of the genus Leishmania and Plasmodium, which are responsible for Leishmaniasis and Malaria, respectively. In addition, their toxicity was evaluated in mammalian cells using murine peritoneal macrophages. Regarding the leishmanicidal activity, part of the compounds presented activity against Leishmania sp, with emphasis on compound 1a in amastigotes of L. amazonensis (IC 50 of 8.1 μM), a close result to the reference drug Miltefosine (IC 50 of 4.15 μM). Most compounds did not present expressive cytotoxicity for macrophages, except for compounds 1b, 1f and 2d. Plasmodium tests are still under review. These results confirm the potential biological of aminoquinoline derivatives and stimulate further studies with this class of molecules for the treatment of parasitic diseases.

Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-05-02T11:30:44Z No. of bitstreams: 1 robertareissoares.pdf: 1821120 bytes, checksum: 37f4636d8bd6ef3623072173760d6105 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-05-12T15:51:49Z (GMT) No. of bitstreams: 1 robertareissoares.pdf: 1821120 bytes, checksum: 37f4636d8bd6ef3623072173760d6105 (MD5) / Made available in DSpace on 2017-05-12T15:51:49Z (GMT). No. of bitstreams: 1 robertareissoares.pdf: 1821120 bytes, checksum: 37f4636d8bd6ef3623072173760d6105 (MD5) Previous issue date: 2013-02-20 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A malária permanece ao longo dos anos como emergência global em saúde pública. São aproximadamente 87 milhões de casos e 106.820 mortes ocorridas anualmente no mundo. As principais estratégias atuais no controle da doença se baseiam no diagnóstico precoce e tratamento oportuno dos casos, devendo ser considerada a resistência dos parasitos a praticamente todos os fármacos atualmente em uso, o que torna urgente a busca por novos antimaláricos. Neste contexto, análogos de 4-aminoquinolinas e 6-mercaptopurinas contendo 1,2,3-triazol ou esteroide foram sintetizados e tiveram sua atividade antimalárica investigada utilizando o teste supressivo de Peters em modelo murino de infecção por Plasmodium berghei NK65. Dentre os 11 análogos avaliados, 5 exibiram atividade antimalárica significativa, alcançando percentuais de supressão de até 81% em relação ao controle não tratado. Além da atividade antimalárica, um composto promissor não pode apresentar efeitos indesejáveis às células do hospedeiro, sendo assim foram avaliadas a citotoxicidade e atividade hemolítica dos análogos. A maioria dos compostos foram considerados seguros às células do hospedeiro mesmo nas maiores concentrações avaliadas. Portanto, estes análogos merecem ser objeto de futuras investigações visando compor novos antimaláricos. / Malaria remains over the years as a global public health emergency. There are approximately 87 million cases and 106.820 deaths worldwide each year. The main current strategies to control the disease are based on early diagnosis and appropriate treatment cases and should be considered the resistance of parasites to almost all drugs currently in use, which makes urgent the search for new antimalarials. In this context, analogs of 4-aminoquinolines and 6-mercaptopurines containing 1,2,3-triazole or steroid were synthesized and investigated for their antimalarial activity using the 4-day suppressive test described by Peters in a murine model of infection by Plasmodium berghei NK65. Among the 11 analogues evaluated, 5 exhibited significant antimalarial activity, reaching percentages of suppression up to 81% compared to untreated control. Besides antimalarial activity, a promising compound cannot have undesirable effects on host cells, thus was evaluated the cytotoxicity and hemolytic activity of analogs. Most compounds were considered safe to host cells even at the highest concentrations evaluated. So, this analogs deserve to be object of future investigations aiming compose new antimalarials.

Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-08-17T11:16:52Z No. of bitstreams: 1 lucianamariaribeiroantinarelli.pdf: 16756474 bytes, checksum: 2304892df326fd4997c78bc7f0870bae (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-08-17T11:34:54Z (GMT) No. of bitstreams: 1 lucianamariaribeiroantinarelli.pdf: 16756474 bytes, checksum: 2304892df326fd4997c78bc7f0870bae (MD5) / Made available in DSpace on 2017-08-17T11:34:54Z (GMT). No. of bitstreams: 1 lucianamariaribeiroantinarelli.pdf: 16756474 bytes, checksum: 2304892df326fd4997c78bc7f0870bae (MD5) Previous issue date: 2017-02-15 / As leishmanioses representam um grande problema de saúde pública mundial com sérias limitações na quimioterapia atual, como: número limitado de fármacos, baixa eficácia, custo elevado, crescente resistência parasitária e toxicidade. O objetivo do presente trabalho foi avaliar a atividade de uma série de dez derivados de 4- aminoquinolinas em espécies de Leishmania relacionadas a leishmaniose tegumentar. Avaliar os possíveis mecanismos de ação do composto com atividade antileishmanial promissora, identificando as organelas alvo e os processos de morte celular desencadeados no parasito, bem como a sua eficácia in vivo. Dentre os compostos avaliados, o derivado AMQ-j foi o mais ativo, com atividade expressiva em L. amazonensis e L. braziliensis (CI50 menor que 6.0 μg/mL e 2,5 μg/mL em promastigotas e amastigotas intracelulares, respectivamente) para ambas as espécies avaliadas. O composto AMQ-j apresentou baixa toxicidade para macrófagos murinos (CC50> 40.0 μg/mL), sendo mais tóxico para amastigotas intracelulares (Índice de Seletividade>12,0). Os resultados preliminares acerca do modo de ação apontam que o composto AMQ-j induziu drásticos efeitos na mitocôndria do parasito e caracterizado pelo colapso do potencial de membrana mitocondrial (ΔΨm), inchaço da organela, aumento na produção de Espécies Reativas do Oxigênio (EROS) e acúmulo de corpúsculos lipídicos (CLs) no citoplasma. O tratamento com AMQ-j induziu nas formas promastigotas uma série de alterações bioquímicas e celulares sugestivas de morte por apoptose-like como redução do volume celular, exposição de fosfatidilserina no folheto externo da membrana plasmática, manutenção da integridade da membrana plasmática e alterações drásticas no núcleo celular evidenciadas por meio da desorganização da cromatina e fragmentação do DNA. O efeito do composto em promastigotas está também associado à indução de morte por autofagia evidenciada pelo aumento de vacúolos autofágicos, presença de corpos multivesiculares dentro de vacúolos, vesículas citoplasmáticas e acúmulo de compartimentos acídicos no citoplasma dos promastigotas. O composto também induziu fragmentação do DNA dos amastigotas intracelulares de modo seletivo, sem induzir fragmentação da célula hospedeira. Estudos in silico sugerem que AMQ-j é um potencial inibidor da tripanotiona redutase (TryR), enzima fundamental na defesa antioxidante do parasito. Estudos in vivo em modelo murino de infecção com L. amazonensis demonstraram a eficácia do AMQ-j pela via intralesional na redução do tamanho da lesão e da carga parasitária, sem indução de toxicidade hepática, cardíaca e renal. Os estudos de predição in silico relacionados a propriedades farmacocinéticas (ADMET) e características físico-químicas (regra de Lipinsky) sugerem que AMQ-j pode ser utilizado pela via oral. O efeito leishmanicida do composto está associado a múltiplos alvos, desencadeando a morte do parasito por diferentes vias, como apoptose e autofagia. O efeito in vivo do composto aponta para a necessidade da continuidade dos estudos no intuito de melhor estabelecer o seu efeito leishmanicida. / Leishmaniasis represents a major global public health problem with serious limitations in current chemotherapy, such as limited number of drugs, low efficacy, high cost, increasing parasitic resistance and toxicity. The objective of the present study was to evaluate a series of ten 4-aminoquinolines derivatives (AMQs) on Leishmania species related to tegumentary leishmaniasis. It was also evaluated the possible mechanisms of action of a compound with promising leishmanicidal activity, identifying the target organelles and the type of death triggered in the parasite, as well as to its leishmanicidal effect in vivo. Among the evaluated compounds, the AMQ-j derivative was the most active, with expressive activity on L. amazonensis and L. braziliensis (IC50 less than 6.0 μg/mL and 2.5 μg/mL against intracellular promastigotes and amastigotes, respectively) for both evaluated species. Furthermore, AMQ-j showed low toxicity for murine macrophages (CC50> 40.0 μg/mL) being more destructive to the intracellular parasites (selectivity index > 12.0). Preliminary studies about the mode of action showed that AMQ-j compound induced marked effects on the parasite mitochondria, characterized by mitochondrial membrane potential collapse (ΔΨm), organelle swelling, increased of Reactive Oxygen Species (ROS) production and lipidic bodies accumulation in the cytoplasm. The treatment with AMQ-j induced in the promastigote forms a series of biochemical and cellular alterations which suggest apoptosis-like death, including reduction of cellular volume, phosphatidylserine exposure on the outer leaflet of the plasma membrane, maintenance of plasma membrane integrity and drastic changes in the cell nucleus evidenced by chromatin disorganization and DNA fragmentation. The effect of AMQ-j in promastigote forms is also associated with the induction of death by autophagy evidenced by the increase of autophagic vacuoles, presence of multivesicular bodies inside vacuoles, cytoplasmic vesicles and accumulation of acidic compartments in the promastigote cytoplasm. The compound also induced DNA fragmentation of intracellular amastigotes selectively, without inducing host cell fragmentation. In silico studies suggest that this compound is a potential inhibitor of key redox enzyme trypanothione reductase (TryR). In vivo studies in murine infection model of L. amazonensis demonstrated the efficacy of AMQ-j by the intralesional route, reducing lesion size and parasite load, without induction of hepatic, cardiac and renal toxicity. The in silico prediction studies on pharmacokinetic properties (ADMET) and physico-chemical characteristics (Lipinsky's rule) suggest that AMQ-j can be used orally. Taken together, the results suggest that the leishmanicidal effect of the compound is associated with multiple targets and triggers parasite death through different pathways, including apoptosis and autophagy. The in vivo effect indicates that studies with this compound should be continued in order to better establish its leishmanicidal effect.